AU2017201572B2

AU2017201572B2 - Targeted/immunomodulatory fusion proteins and methods for making same

Info

Publication number: AU2017201572B2
Application number: AU2017201572A
Authority: AU
Inventors: Nagaraj Govindappa; Kedarnath Sastry; Melina Maria Soares
Original assignee: Biocon Ltd
Current assignee: Biocon Ltd
Priority date: 2012-04-30
Filing date: 2017-03-07
Publication date: 2018-09-27
Anticipated expiration: 2033-03-13
Also published as: PT2844667T; HK1202295A1; AU2013255542A1; JP2015516815A; NZ701821A; US12049632B2; CN104334573A; JP2020138977A; CN108503711A; US9758582B2; DK3489254T3; ES2938182T3; US9340617B2; US20180066057A1; JP7035115B2; CA3212759A1; JP7307218B2; PL3489254T3; CN110563850A; CA3004695C

Abstract

OF THE INVENTION The present invention relates generally to the field of generating fusion proteins to be used in cancer therapy, and more specifically, to nucleotide sequences encoding the fusion proteins, wherein the chimeric fusion proteins comprises at least one targeting moiety and at least one immunomodulatory moiety that counteracts the immune tolerance of cancer cells. /1 1)

Description

The present invention relates generally to the field of generating fusion proteins to be used in cancer therapy, and more specifically, to nucleotide sequences encoding the fusion proteins, wherein the chimeric fusion proteins comprises at least one targeting moiety and at least one immunomodulatory moiety that counteracts the immune tolerance of cancer cells.

2017201572 07 Mar 2017

ABSTRACT OF THE INVENTION

2017201572 07 Mar 2017

TARGETED/IMMUNOMODULATORY FUSION PROTEINS AND METHODS FOR

MAKING SAME

CROSS REFERENCE TO RELATED APPLICATIONS [001] The application claims priority to Indian Patent Application No. 1689/CHE/2012 filed on April 30, 2012 and Indian Patent Application No. 1690/CHE/2012 filed on April 30, 2012 and claims the benefit of Australian patent application 2013255542 filed 13 March 2013, the contents of each of which are hereby incorporated by reference herein for all purposes.

BACKGROUND OF THE INVENTION [002] Technical Field [003] The present invention relates generally to the field of generating fusion proteins to be used in cancer therapy, and more specifically, to nucleotide sequences encoding the fusion proteins, wherein the fusion or chimeric polypeptides comprises at least one targeting moiety and at least one immunomodulatory moiety that counteracts the immune tolerance of cancer cells.

[004] Related Art [005] The immune system provides the human body with a means to recognize and defend itself against microorganisms and substances recognized as foreign or potentially harmful. While passive immunotherapy of cancer with monoclonal antibodies and passive transfer of T cells to attack tumor cells have demonstrated clinical efficacy, the goal of active therapeutic vaccination to induce these immune effectors and establish immunological memory against tumor cells has remained challenging. Several tumor-specific and tumorassociated antigens have been identified, yet these antigens are generally weakly immunogenic and tumors employ diverse mechanisms to create a tolerogenic environment that allows them to evade immunologic attack. Strategies to overcome such immune tolerance and activating robust levels of antibody and/or T cell responses hold the key to effective cancer immunotherapy. More important, the individual proteins and how to create an active chimeric polypeptide with an active tertiary structure needs to be explored.

2017201572 07 Mar 2017

SUMMARY OF THE INVENTION [006] The present invention provides polynucleotides, as well as polypeptides encoded thereby, that are expressed in cancer cells. These polynucleotides and expressed polypeptides are useful in a variety of therapeutic methods for the treatment of cancer. The present invention further provides methods of reducing growth of cancer cells by counteracting immune tolerance of cancer cells, wherein T cell remain active and inhibit the recruitment of T-regulatory that are known to suppress the immune system’s response to the tumor. Thus the chimeric polypeptides generated by the polynucleotides sequences of the present invention are useful for treating cancer because of the expressed fusion or chimeric polypeptides.

[007] In one aspect, the present invention provides for chimeric polypeptides containing at least one targeting moiety to target a cancer cell and at least one immunomodulating moiety that counteracts immune tolerance of cancer cell, wherein the targeting moiety and the immunomodulating moiety are linked by a amino acid spacer of sufficient length of amino acid residues so that both moieties can successfully bond to their individual target. In the alternative, the targeting moiety and the immunomodulating moiety that counteract immune tolerance of cancer cell may be bound directly to each other. The chimeric/fusion polypeptides of the invention are useful for binding to a cancer cell receptor and reducing the ability of cancer cells to avoid an immune response.

[008] The present invention is based on preparing chimeric/fusion proteins by expression of polynucleotides encoding the fusion proteins that counteract or reverse immune tolerance of cancer cells. Cancer cells are able to escape elimination by chemotherapeutic agents or tumor-targeted antibodies via specific immunosuppressive mechanisms in the tumor microenvironment and such ability of cancer cells is recognized as immune tolerance. Such immunosuppressive mechanisms include immunosuppressive cytokines (for example, Transforming growth factor beta (TGF-β)) and regulatory T cells and/or immunosuppressive myeloid dendritic cells (DCs). By counteracting tumor-induced immune tolerance, the present invention provides effective compositions and methods for cancer treatment, optional in combination with another existing cancer treatment. The present invention provides strategies to counteract tumor-induced immune tolerance and enhance the antitumor efficacy

2017201572 07 Mar 2017 of chemotherapy by activating and leveraging T cell-mediated adaptive antitumor against resistant or disseminated cancer cells.

[009] In another aspect, the present invention provides a molecule including at least one targeting moiety fused with at least one immunomodulatory moiety. The targeting moiety specifically binds a target molecule, and the immunomodulatory moiety specifically binds one of the following molecules: (i) Transforming growth factor-beta (TGF-β): (ii) Programmed death- 1 ligand 1 (PD-L1) or Programmed death- 1 ligand 2 (PD-L2); (iii) Receptor activator of nuclear factor-KB (RANK) ligand (RANKL); (iv) Transforming growth factor-beta receptor (TGF-pR); (v) Programmed death-1 (PD-1 ); (vi) 4-1BB receptor or (vii) Receptor activator of nuclear factor-κΒ (RANK).

[0010] In a further aspect, the targeting moiety includes an antibody, antibody fragment including the light or heavy chains of the antibody, scFv, or Fc-containing polypeptide that specifically binds a component of a tumor cell, tumor antigen, tumor vasculature, tumor microenvironment, or tumor-infiltrating immune cell. Preferably, the targeting moiety is an antibody or a fragment thereof having binding affinity for a component on a tumor cell. Notably each of the heavy chain and light chain may individually be linked to a separate and distinct immunomodulatory moiety. Further, a heavy or light chain of an antibody targeting moiety may be linked to an immunomodulatory moiety which in turn can be further linked to a second immunomodulatory moiety wherein there is a linker between the two immunomodulatory moieties.

[0011] In a still further aspect, there is provided a chimeric polypeptide that comprised a tumor targeting moiety and an immunomodulatory moiety comprising a molecule that binds transforming growth factor beta (TGF-β), wherein the tumor targeting moiety is an antibody that binds to EGFR1, where in the antibody can be the full antibody, heavy chain or light chain. The tumor targeting moiety may include monoclonal antibodies that target a cancer cell, including but not limited to cetuximab, trastuzumab, ritubximab, ipilimumab, tremelimumab, muromonab-CD3, abciximab, daclizumab, basiliximab, palivizumab, infliximab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, omalizumab, tositumomab, 1-131 tositumomab, efalizumab, bevacizumab, panitumumab, pertuzumab, natalizumab, etanercept, IGN101 (Aphton), volociximab (Biogen Idee and PDL BioPharm), Anti-CD80 mAh (Biogen Idee), Anti-CD23 mAh (Biogen Idel), CAT-3888

2017201572 07 Mar 2017 (Cambridge Antibody Technology), CDP-791 (Imclone), eraptuzumab (Immunomedics), MDX-010 (Medarex and BMS), MDX-060 (Medarex), MDX-070 (Medarex), matuzumab (Merck), CP-675,206 (Pfizer), CAL (Roche), SGN-30 (Seattle Genetics), zanolimumab (Serono and Genmab), adecatumumab (Sereno), oregovomab (United Therapeutics), nimotuzumab (YM Bioscience), ABT-874 (Abbott Laboratories), denosumab (Amgen), AM 108 (Amgen), AMG 714 (Amgen), fontolizumab (Biogen Idee and PDL BioPharm), daclizumab (Biogent Idee and PDL BioPharm), gobmumab (Centocor and Schering-Plough), CNTO 1275 (Centocor), ocrelizumab (Genetech and Roche), HuMax-CD20 (Genmab), belimumab (HGS and GSK), epratuzumab (Immunomedics), MLN1202 (Millennium Pharmaceuticals), visilizumab (PDL BioPharm), tocilizumab (Roche), ocrerlizumab (Roche), certolizumab pegol (UCB, formerly Celltech), eculizumab (Alexion Pharmaceuticals), pexelizumab (Alexion Pharmaceuticals and Procter & Gamble), abeiximab (Centocor), ranibizimumab (Genetech), mepolizumab (GSK), TNX-355 (Tanox), or MYO-029 (Wyeth).

[0012] In an another aspect, the tumor targeting moiety is a monoclonal antibody that binds to HER2/Neu, CD20, CTLA4, EGFR1 and wherein the antibody can be the full antibody, heavy chain or light chain.

[0013] In yet another aspect, the targeting moiety is a molecule that specifically binds epidermal growth factor receptor (EGFR1, Erb-B 1), HER2/neu (Erb-B2), CD20, cytotoxic Tlymphocyte antigen-4 (CTLA-4) which is essential for Treg function (CD 152); H-land Interleukin- 6 (IL-6).

[0014] In a still further aspect, the targeting moiety specifically binds a component of a regulatory T cell (treg), myeloid suppressor cell, or dendritic cell. In another aspect, the targeting moiety specifically binds one of the following molecules: (i) CD4; (ii) CD25 (IL2ct receptor; IL-2aR); (iii) Transforming growth factor-beta receptor (TGF-pR); (vi) Transforming growth factor-beta (TGF-β): (vii) Programmed Death- 1 (PD-1); (viii) Programmed death- 1 ligand (PD-LI or PD-L2.

[0015] In another aspect, the immunomodulatory moiety specifically binds one of the following molecules: (i) Transforming growth factor-beta (TGF-β): (ii) Programmed death-1 ligand (PD-LI or PD-L2); or 4-IBB receptor.

2017201572 07 Mar 2017 [0016] In yet another aspect, the immunomodulatory moiety includes a molecule that binds TGF-β and inhibits the function thereof. Specifically the immunomodulatory moiety includes an extracellular ligand-binding domain of Transforming growth factor-beta receptor TGF[iRII, TGF-[iRIIb, or TGF-[iRIII. In another aspect the immunomodulatory moiety includes an extracellular ligand-binding domain (ECD) of TGF-[iRII. Still further the immunomodulatory moiety may include H-4-1BB ligand which binds to the 4-1BB receptor to stimulate T-cells to help eradiate tumor.

[0017] In a still further aspect, the targeting moiety includes an antibody, antibody fragment, or polypeptide that specifically binds to HER2/neu, EGFR1, CD20, or cytotoxic Tlymphocyte antigen-4 (CTLA-4) and wherein the immunomodulatory moiety includes an extracellular ligand-binding domain of TGF-3RII.

[0018] In yet another aspect, the immunomodulatory moiety includes a molecule that specifically binds to and inhibit the activity of Programmed death- 1 ligand 1 (PD-L 1) or Programmed death- 1 ligand 2 (PD-L2). In another aspect, the immunomodulatory moiety includes an extracellular ligand-binding domain or ectodomain of Programmed Death- 1 (PD1).

[0019] In a further aspect, the targeting moiety includes an antibody, antibody fragment, or polypeptide that specifically binds to HER2/neu, EGFR1, CD20, cytotoxic T-lymphocyte antigen-4 (CTLA-4), CD25 (lL-2a receptor; IL-2aR), or CD4 and wherein, the immunomodulatory moiety includes an extracellular ligand-binding domain or ectodomain of Programmed Death- 1 (PD-1).

[0020] In a still further aspect, the targeting moiety includes an antibody or antibody fragment that specifically binds to CD20, and the immunomodulatory moiety includes a sequence from transforming growth factor-β (TGF-β).

[0021] In one aspect, the present invention provides for optimized genes encoding for a fusion polypeptide comprising at least one targeting moiety and at least one immunomodulatory moiety for treating cancer in a human subject wherein the optimized genes have been modified to increase expression in a human subject, preferably the optimized

2017201572 07 Mar 2017 genes comprise sequences for encoding a targeting moiety or an immunomodulatory moiety selected from SEQ ID NOs: 12 to 28.

[0022] In another aspect, the present invention provides for a vector comprising optimized genes for treating cancer in a human subject wherein the optimized genes have been modified to increase CG sequences. Preferably, the vector includes sequences for encoding at least one targeting moiety and at least one immunomodulatory moiety selected from SEQ ID NOs: 12 to 28.

[0023] In yet another aspect, the present invention provides for a method of treating cancer in a subject, the method comprising:

a. providing at least one recombinant vector comprising nucleotide sequences that encode at least one targeting moiety and at least one immunomodulatory moiety selected from SEQ ID NOs: 12 to 28; and

b. administering the recombinant vector to the subject under conditions such that said nucleotide sequences are expressed at a level which produces a therapeutically effective amount of the encoded fusion proteins in the subject.

[0024] In an alternative aspect, the present invention provides an expression vector comprising polynucleotides of optimized genes that encode at least one targeting moiety and at least one immunomodulatory moiety selected from SEQ ID NOs: 12 to 28.

[0025] In yet another aspect, the present invention provides a recombinant host cell transfected with a polynucleotide that encodes a fusion protein peptide of the present invention.

[0026] In a still further aspect, the present invention contemplates a process of preparing a fusion protein of the present invention comprising:

a. transfecting a host cell with polynucleotide sequences that encode chimeric fusion proteins to produce a transformed host cell, wherein the polynucleotide sequences encode at least one targeting moiety and at least one immunomodulatory moiety selected from SEQ ID NOs: 12 to 28; and

b. maintaining the transformed host cell under biological conditions sufficient for expression of the peptide.

[0027] In another aspect, the present invention relates to the use of a chimeric fusion protein, as shown in Figures 1 to 15, in the use of a medicament for the treatment of cancer.

Preferably, the fusion protein is expressed in a host cell and such expressed proteins are administered in a therapeutic amount to reduce the effects of cancer in a subject in need thereof.

2017201572 07 Mar 2017 [0028] In a still further aspect, the present invention provides a method of preventing or treating a neoplastic disease. The method includes administration to a subject in need thereof one or more fusion proteins of the invention, in various aspects, the subject is administered one or more molecule of the invention in combination with another anticancer therapy, in one aspect, the anticancer therapy includes a chemotherapeutic molecule, antibody, small molecule kinase inhibitor, hormonal agent or cytotoxic agent. The anticancer therapy may also include ionizing radiation, ultraviolet radiation, cryoablation, thermal ablation, or radio frequency ablation.

[0029] In yet another aspect, the present invention provides for a method of preparing therapeutically active antibody-peptide fusion proteins, the method comprising;

a. preparing a codon optimized sequence of the said fusion protein;

b. cloning the optimized sequence of said fusion protein in a host cell capable of transient or continued expression;

c. growing the host cell in a media under suitable conditions for growing and allowing the host cell to express the cloned protein; and

d. subjecting the expressed protein to purification and optionally checking the bispecific binding capabilities of the protein to its targets.

[0030] In a preferred embodiment the therapeutically active antibody-peptide fusion proteins is a targeting antibody fused to one or more immunomodulating moiety that counteracts immune tolerance of a cancer cell. In one aspect, the immunomodulating moiety may be linked by an amino acid spacer of sufficient length to allow bi-specific binding of the molecule. The immunomodulating moiety may be bound to either the C-terminus of the heavy or light chain of the antibody [0031] In a preferred method as described above, the immunomodulating moiety is (i) Transforming growth factor-beta (TGF-β), (ii) Programmed death-1 (PD-1 ), (iii) CTLA-4 or

2017201572 07 Mar 2017 (iv) 4-IBB or parts thereof and the targeting antibody binds epidermal growth factor receptor (EGFR1, Erb-B 1), HER2/neu (Erb-B2), CD20, CD6, CTLA-4, Mucin l(MUC-l),

Interleukin-2 (IL-2) or Interleukin- 6 (IL-6).

[0032] The method of the present invention provides nucleotide sequences that encode the therapeutically active antibody-peptide fusion proteins and such expression may be conducted in a transient cell line or a stable cell line. The transient expression is accomplished by transfecting or transforming the host with vectors carrying the fusion proteins into mammalian host cells [0033] Once the fusion peptides are expressed, they are preferably subjected to purification and in-vitro tests to check its bi-specificity, that being, having the ability to bind to both the target moiety and immunomodulating moiety. Such tests may include in-vitro test such as ELISA or NK/T-cell binding assays to validate bi-functional target binding or immune cell stimulation.

[0034] Notably once the specific fusion peptides demonstrate the desired bi-specificity, such fusion peptides are selected for sub-cloning into a stable cell line for larger scale expression and purification. Such stable cell lines are previously disclosed, such as a mammalian cell line, including but not limited to HEK293, CHO or NSO.

[0035] In a further aspect, the culture medium can be improved by additions to such medium. For example, the culture medium may include a divalent transitional metallic salt which is added to the cell culture either initially or in fed-batch mode to reduce accumulation of lactate during culturing and/or reduce heterogeneity of the fusion proteins. A desirable transitional metallic salt includes a zinc ion and the addition of the metal ion may be carried out during different phases of the production.

[0036] Other features and advantages of the invention will be apparent from the following detailed description, drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

2017201572 07 Mar 2017 [0037] Figure 1 shows the amino acid sequences of with the amino acid sequence of AntiHER2/neu-TGF3RII fusion protein at LC constant region with the amino acid sequence of anti-HER2/neu heavy chain (SEQ ID NO: 1) and anti-HER2/neu light chain (SEQ ID NO: 2) attached to amino residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters and wherein a linker (SEQ ID NO: 3) is positioned between the antiHER2/neu light chain and TGF-3RII and shown in italics.

[0038] Figure 2 shows the amino acid sequences of Anti-EGFRl-TGFpRII fusion protein at LC constant region with amino acid sequence of Anti-EGFRl heavy chain (SEQ ID NO: 5) and the amino acid sequence of Anti-EGFRl light chain (SEQ ID NO: 6) attached to amino acid residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters and wherein a linker (SEQ ID NO: 3) is positioned between the Anti-EGFRl light chain and TGF-3RII and shown in italics.

[0039] Figure 3 shows the amino acid sequences of Anti-CTLA4-TGF3RII fusion protein at LC constant region with amino acid sequence of anti-CTLA4 heavy chain (SEQ ID NO: 7) and amino acid sequence of anti-CTLA4 light chain (SEQ ID NO: 8) attached to amino acid residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters and wherein a linker (SEQ ID NO: 3) is positioned between the anti-CTLA4 light chain and TGF-3RII and shown in italics.

[0040] Figure 4 shows the amino acid sequences of Anti-HER2/neu HC-4-1BB and LCTGFpRII fusion protein with amino acid sequence of Anti-HER2/neu/HC-4-lBB fusion protein wherein the amino acid sequence for Anti-HER2/neu heavy chain (SEQ ID NO: 1) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for 4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text font and amino acid sequence of anti-HER2/neu light chain (SEQ ID NO: 2) attached to amino residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters and wherein a linker (SEQ ID NO: 3) is positioned between the anti-HER2/neu light chain and TGF-3RII and shown in italics.

[0041] Figure 5 shows the amino acid sequence of Anti-EGFRl HC-4-1BB and LC-TGF3RII fusion protein with amino acid sequence of Anti-EGFRl heavy chain-4-IBB fusion protein wherein the amino acid sequence for Anti-EGFRl heavy chain (SEQ ID NO: 5) is attached to

2017201572 07 Mar 2017 a linker (SEQ ID NO: 3) is shown in italics and the sequence for 4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text font and amino acid sequence of light chain AntiEGFR1 (SEQ ID NO: 6) attached to amino residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) therebetween.

[0042] Figure 6 shows the amino acid sequence of Anti-CTLA4 HC-4-1BB and LCTGFpRII fusion protein with amino acid sequence of Anti-CTLA4 heavy chain-4-IBB fusion protein wherein the amino acid sequence for Anti-CTLA4 heavy chain (SEQ ID NO: 7) is attached to a linker (SEQ ID NO: 3) is shown in italics and the sequence for 4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text font and amino acid sequence of Anti-CTLA4 light chain (SEQ ID NO: 8) is attached to amino residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) therebetween.

[0043] Figure 7 shows the amino acid sequence of Anti-HER2/neu HC-PD1 and LCTGFpRII fusion protein with amino acid sequence of Anti-HER2/neu heavy chain-PDl fusion protein wherein the amino acid sequence for the Anti-HER2/neu heavy chain (SEQ ID NO: 1) is attached to a linker (SEQ ID NO: 3) is shown in italics and the sequence for PD1 (immunomodulatory moiety) (SEQ ID NO: 10) is in written text font and amino acid sequence of Anti-HER2/neu light chain (SEQ ID NO: 2) is attached to amino residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) therebetween.

[0044] Figure 8 shows the amino acid sequence of Anti-EGFRl HC-PD1 and LC-TGF3RII fusion protein with amino acid sequence of Anti-EGFRl heavy chain-PDl fusion protein wherein the amino acid sequence Anti-EGFRl heavy chain (SEQ ID NO: 5) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for PD1 (immunomodulatory moiety) (SEQ ID NO: 10) is in written text font and amino acid sequence of Anti-EGFRl light chain (SEQ ID NO: 6) attached to amino residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) therebetween.

2017201572 07 Mar 2017 [0045] Figure 9 shows the amino acid sequence of Anti-CTLA4 HC-PD1 and LC-TGF3RII fusion protein with amino acid sequence of Anti-CTLA4 heavy chain-PDl fusion protein wherein the amino acid sequence Anti-CTLA4 heavy chain (SEQ ID NO: 7) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for PD1 (immunomodulatory moiety) (SEQ ID NO: 10) is in written text font and amino acid sequence of Anti-CTLA4 light chain (SEQ ID NO: 8) attached to amino residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) therebetween.

[0046] Figure 10 shows the amino acid sequence of Anti-HER2/neu HC-TGF3RII-4-lBB fusion protein with amino acid sequence of Anti-HER2/neu heavy chain-TGF3RII-4-lBB fusion protein wherein the amino acid sequence for Anti-HER2/neu heavy chain (SEQ ID NO: 37) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFpRII (immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for 4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text font with linker between (SEQ ID No: 11) and including the amino acid sequence of Anti-HER2/neu light chain (SEQ ID NO: 2).

[0047] Figure 11 shows the amino acid sequence of Anti-EGFRl HC-TGF3RII-4-lBB fusion protein with amino acid sequence of Anti-EGFRl heavy chain-TGF3RII-4-lBB fusion protein wherein the amino acid sequence for Anti-EGFRl heavy chain (SEQ ID NO: 38) sequence is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFpRII (immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for 4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text font with linker between (SEQ ID NO: 11) and including the amino acid sequence of Anti-EGFRl light chain (SEQ ID NO: 6).

[0048] Figure 12 shows the amino acid sequence of Anti-CTLA4 HC-TGF3RII-4-lBB fusion protein with amino acid sequence of Anti-CTLA4 heavy chain-TGF3RII-4-lBB fusion protein wherein the amino acid sequence Anti-CTLA4 heavy chain (SEQ ID NO: 39) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFpRII (immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for 4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text font with

2017201572 07 Mar 2017 linker between (SEQ ID NO: 11) and including the amino acid sequence of Anti-CTLA4 light chain (SEQ ID NO: 8).

[0049] Figure 13 shows the amino acid sequence of Anti-HER2/neu HC-TGF3RII-PD1 fusion protein with amino acid sequence of Anti-HER2/neu heavy chain-TGF3RII-PDl fusion protein wherein the amino acid sequence Anti-HER2/neu heavy chain (SEQ ID NO: 37) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFpRII (immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for PD-1 (immunomodulatory moiety) (SEQ ID NO: 10) is in written text font with linker between (SEQ ID No: 11) and including the amino acid sequence of Anti-HER2/neu light chain (SEQ ID NO: 2).

[0050] Figure 14 shows the amino acid sequence of Anti-EGFRl HC-TGF3RII-PD1 fusion protein with amino acid sequence of Anti-EGFRl heavy chain-TGF3RII-PDl fusion protein wherein the amino acid sequence Anti-EGFRl heavy chain (SEQ ID NO: 38) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFpRII (immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for PD-1 (immunomodulatory moiety) (SEQ ID NO: 10) is in written text font with linker between (SEQ ID No: 11) and including the amino acid sequence of Anti-EGFRl light chain (SEQ ID NO: 6).

[0051] Figure 15 shows the of Anti-CTLA4 HC-TGF3RII-PD1 fusion protein with amino acid sequence of Anti-CTLA4 heavy chain-TGF3RII-PDl fusion protein wherein the amino acid sequence Anti-CTLA4 heavy chain (SEQ ID NO: 39) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFpRII (immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for PD-1 (immunomodulatory moiety) (SEQ ID NO: 10) is in written text font with linker between (SEQ ID NO: 11) and including the amino acid sequence of Anti-CTLA4 light chain (SEQ ID NO: 8).

[0052] Figure 16 shows the nucleotide sequence of Anti-HER2/neu heavy chain constant region with linker (SEQ ID NO: 12) and TGFpRII ECD (SEQ ID NO: 13) that have been codon optimized for expression in CHO cell.

2017201572 07 Mar 2017 [0053] Figure 17 shows the nucleotide sequence of Anti-HER2/neu heavy chain variable region (SEQ ID NO: 14), Anti-HER2/neu light chain variable region (SEQ ID NO: 15) and

Anti-EGFRl heavy chain constant region with linker (SEQ ID NO: 16) that have been codon optimized for expression in CHO cell.

[0054] Figure 18 shows the nucleotide sequence of Anti-EGFRl heavy chain variable region (SEQ ID NO: 17), Anti-EGFRl light chain variable region (SEQ ID NO: 18), Anti-CTLA4 heavy chain variable region (SEQ ID NO: 19) and Anti-CTLA4 light chain variable region (SEQ ID NO: 20) that have been codon optimized for expression in CHO cell.

[0055] Figure 19 shows the nucleotide sequence of Anti CD20 IgGl molecule (SEQ ID NO: 21), Anti-CD20 heavy chain variable region (SEQ ID NO: 22) and Anti-CD20 light chain variable region (SEQ ID NO: 23) that have been codon optimized for expression in CHO cell.

[0056] Figure 20 shows the nucleotide sequence of 4-1BB (SEQ ID NO: 24) and Anti-IL6R heavy chain (SEQ ID NO: 25) that have been codon optimized for expression in CHO cell.

[0057] Figure 21 shows the nucleotide sequence of Anti-IL6R light chain variable region (SEQ ID NO: 26), Anti-4-lBB heavy chain (SEQ ID NO: 27) and Anti-4-lBB light chain variable region (SEQ ID NO: 28) that have been codon optimized for expression in CHO cell.

[0058] Figure 22 shows the analysis of Protein A purified Anit-HER2/neu-TGF3RII and Anti-EGFRl- TGFpRII at 12 % PAGE [0059] Figure 23 A shows Anti-HER2/neu-TGF3RII samples analyzed by Protein A/SEC Chromtography and B Anti-EGFRl-TGFpRII samples analyzed by Protein A/SEC Chromtography.

[0060] Figure 24 A shows that Anti-HER2/neu-TGF3RII and Anti-EGFRl-TGFpRII molecules bind to the TGF3 indicating that the fusion protein is functional and B shows that Anti-HER2-TGF3RII inhibits the proliferation of BT474 cell line similar to the Bmab200 (Herceptin).

[0061] Figure 25 shows that Anti-EGFRl-TGFpRII-inhibits the proliferation of A431 cell line similar to the Cetuximab.

2017201572 07 Mar 2017 [0062] Figure 26 shows the ADCC activity of Anti-HER2-TGF3RII on BT474 cells is similar to that of Bmab200 (Herceptin).

[0063] Figure 27 shows the ADCC activity of Anti-EGFRl-TGFpRII on A431 cells wherein the ADCC activities are similar to that of Cetuximab.

[0064] Figure 28 shows the ADCC activity of ADCC activity of Anti-EGFRl-4-lBB in comparison with Anti-EGFR l -TGFpRIIand cetuximab.

[0065] Figure 29 A shows that the binding activity of Anti-CTLA4-TGF3RII to TGFpi is comparable to Anti-EGFR1-TGF3RII and B shows that the binding activity of Anti-CTLA4TGFpRII to CTLA4.

[0066] Figure 30 A shows the binding activity of Anti-CTLA4-TGF3RII to determine the level of PDl-Fc binding and B shows the binding activity of Anti-EGRFl-4-lBB to determine the binding of 4-1BBL.

[0067] Figure 31 shows the binding activity of Anti-EGFRl-4-lBB to EGFR and B shows the binding activity of PDl-Fc-4-lBB to find out PDLl-Fc.

[0068] Figure 32 shows the binding activity of Anti-EGFRl-PDl to EGFR and PD1.

[0069] Figure 33 shows photographs of expressed proteins and reduction alkylation thereof.

[0070] Figure 34 A shows the mass spectrum Mass Spectrum of light chain (LC) (Reduced) of Anti-HER2/neu-TGF3RII ECD fusion and B shows Deconvoluted Mass Spectrum of LC (Reduced) of Anti-HER2/neu-TGF3RII ECD fusion.

[0071] Figure 35 shows the Mass Spectrum of heavy chain (HC) (Reduced) of AntiHER2/neu-TGF3RII ECD fusion.

2017201572 07 Mar 2017 [0072] Figure 36A shows the Mass Spectrum of LC (Reduced) of Anti-EGFRl-TGFpRII

ECD and B shows the Deconvoluted Mass Spectrum of LC (Reduced) of Anti-EGFRlTGFpRII ECD.

[0073] Figure 37 shows the Mass Spectrum of HC (Reduced) of Anti-EGFR1-TGF3RII ECD.

[0074] Figure 38 A shows the UV Chromatogram of Tryptic Peptides of Anti-HER2/neuTGFpRII ECD fusion protein and B shows the Total Ion Chromatogram (TIC) of Tryptic Peptides of Anti-HER2/neu-TGF3RII ECD fusion protein.

[0075] Figures 39, 40 and 41 provide lists of expected/observed tryptic peptide of the light chain, heavy chain and linked motif of the Anti-HER2/neu-TGF3RII ECD fusion protein, respectively.

[0076] Figure 42 A shows the UV Chromatogram of Tryptic Peptides of Anti-EGFRlTGFpRII ECD fusion protein and B shows the Total Ion Chromatogram (TIC) of Tryptic Peptides of Anti-EGFR1-TGF3RII ECD fusion protein.

[0077] Figure 43 provides a list of expected/observed tryptic peptide of the light chain of the Anti-EGFRl-TGFpRII ECD fusion protein.

[0078] Figure 44 shows the list of expected/observed tryptic peptide of the heavy chain of the Anti-EGFRl-TGFpRII ECD fusion protein.

[0079] Figure 45 shows the list of expected/observed tryptic peptide of the heavy chain of the Anti-EGFRl-TGFpRII ECD fusion protein.

[0080] Figure 46 shows the amino acid sequences of Cantuzumab -TGFpRII fusion protein at LC constant region with amino acid sequence of Cantuzumab heavy chain (SEQ ID NO: 29) and amino acid sequence of Cantuzumab light chain (SEQ ID NO: 30) attached to amino acid residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters and wherein a linker (SEQ ID NO: 3) is positioned between the Cantuzumab light chain and TGF-3RII and shown in italics.

2017201572 07 Mar 2017 [0081] Figure 47 shows the amino acid sequences of Cixutumumab-TGFpRII fusion protein at LC constant region with amino acid sequence of Cixutumumab heavy chain (SEQ ID NO: 31) and amino acid sequence of Cixutumumab light chain (SEQ ID NO: 32) attached to amino acid residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters and wherein a linker (SEQ ID NO: 3) is positioned between the Cixutumumab light chain and TGF-3RII and shown in italics.

[0082] Figure 48 shows the amino acid sequences of Clivatuzumab-TGFpRII fusion protein at LC constant region with amino acid sequence of Clivatuzumab heavy chain (SEQ ID NO: 33) and amino acid sequence of Clivatuzumab light chain (SEQ ID NO: 34) attached to amino acid residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters and wherein a linker (SEQ ID NO: 3) is positioned between the Clivatuzumab light chain and TGF-3RII and shown in italics.

[0083] Figure 49 shows the amino acid sequences of Pritumumab-TGFpRII fusion protein at LC constant region with amino acid sequence of Pritumumab heavy chain (SEQ ID NO: 35) and amino acid sequence of Pritumumab light chain (SEQ ID NO: 36) attached to amino acid residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters and wherein a linker (SEQ ID NO: 3) is positioned between the Pritumumab light chain and TGF-3RII and shown in italics.

[0084] Figure 50 shows the amino acid sequence of Cantuzumab HC-4-1BB and LCTGFpRII fusion protein wherein the amino acid sequence for the Cantuzumab heavy chain (SEQ ID NO: 29) is attached to a linker (SEQ ID NO: 3) which is shown in italics and the sequence for 4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text font and amino acid sequence of Cantuzumab light chain (SEQ ID NO: 30) is attached to amino residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) therebetween.

[0085] Figure 51 shows the amino acid sequence of Cixutumumab HC-4-1BB and LCTGFpRII fusion protein wherein the amino acid sequence for the Cixutumumab heavy chain (SEQ ID NO: 31) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for 4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text font and amino

2017201572 07 Mar 2017 acid sequence of Cixutumumab light chain (SEQ ID NO: 32) is attached to amino residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) therebetween.

[0086] Figure 52 shows the amino acid sequence of Clivatuzumab HC-4-1BB and LCTGFpRII fusion protein wherein the amino acid sequence for the Clivatuzumab heavy chain (SEQ ID NO: 33) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for 4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text font and amino acid sequence of Clivatuzumab light chain (SEQ ID NO: 34) is attached to amino residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) therebetween.

[0087] Figure 53 shows the amino acid sequence of Pritumumab HC-4-1BB and LCTGFpRII fusion protein wherein the amino acid sequence for the Pritumumab heavy chain (SEQ ID NO: 35) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for 4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text font and amino acid sequence of Pritumumab light chain (SEQ ID NO: 36) is attached to amino residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) therebetween.

[0088] Figure 54 shows the amino acid sequence of Cantuzumab - HC-PD1 and LCTGFpRII fusion protein wherein the amino acid sequence for the Cantuzumab heavy chain (SEQ ID NO: 29) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for PD1 (immunomodulatory moiety) (SEQ ID NO: 10) is in written text font and amino acid sequence of Cantuzumab light chain (SEQ ID NO: 30) is attached to amino residues for TGF3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) therebetween.

[0089] Figure 55 shows the amino acid sequence of Cixutumumab - HC-PD1 and LCTGFpRII fusion protein wherein the amino acid sequence for the Cixutumumab heavy chain (SEQ ID NO: 31) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for PD1 (immunomodulatory moiety) (SEQ ID NO: 10) is in written text font and amino acid sequence of Cixutumumab light chain (SEQ ID NO: 32) is attached to amino residues for

2017201572 07 Mar 2017

TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) therebetween.

[0090] Figure 56 shows the amino acid sequence of Clivatuzumab - HC-PD1 and LCTGFpRII fusion protein wherein the amino acid sequence for the Clivatuzumab heavy chain (SEQ ID NO: 33) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for PD1 (immunomodulatory moiety) (SEQ ID NO: 10) is in written text font and amino acid sequence of Clivatuzumab light chain (SEQ ID NO: 34) is attached to amino residues for TGF-3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) therebetween.

[0091] Figure 57 shows the amino acid sequence of Pritumumab - HC-PD1 and LC-TGF3RII fusion protein wherein the amino acid sequence for the Pritumumab heavy chain (SEQ ID NO: 35) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for PD1 (immunomodulatory moiety) (SEQ ID NO: 10) is in written text font and amino acid sequence of Pritumumab light chain (SEQ ID NO: 36) is attached to amino residues for TGF3RII (immunomodulatory moiety) (SEQ ID NO: 4) identified in bold letters with a linker (SEQ ID NO: 3) therebetween.

[0092] Figure 58 shows the amino acid sequence of Cantuzumab HC-TGF3RII-4-lBB fusion protein wherein the amino acid sequence for Cantuzumab heavy chain (SEQ ID NO: 29) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFpRII (immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for 4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text font with linker between (SEQ ID No: 11) and including the amino acid sequence of Cantuzumab light chain (SEQ ID NO: 30).

[0093] Figure 59 shows the amino acid sequence of Cixutumumab HC-TGF3RII-4-lBB fusion protein wherein the amino acid sequence for Cixutumumab heavy chain (SEQ ID NO: 31) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFpRII (immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for 4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text font with linker between (SEQ ID No: 11) and including the amino acid sequence of Cixutumumab light chain (SEQ ID NO: 32).

2017201572 07 Mar 2017 [0094] Figure 60 shows the amino acid sequence of Clivatuzumab HC-TGF3RII-4-lBB fusion protein wherein the amino acid sequence for Clivatuzumab heavy chain (SEQ ID NO: 33) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFpRII (immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for 4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text font with linker between (SEQ ID No: 11) and including the amino acid sequence of Clivatuzumab light chain (SEQ ID NO: 34).

[0095] Figure 61 shows the amino acid sequence of Pritumumab HC-TGF3RII-4-lBB fusion protein wherein the amino acid sequence for Pritumumab heavy chain (SEQ ID NO: 35) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFpRII (immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for 4-1BB (immunomodulatory moiety) (SEQ ID NO: 9) is in written text font with linker between (SEQ ID No: 11) and including the amino acid sequence of Pritumumab light chain (SEQ ID NO: 36).

[0096] Figure 62 shows the amino acid sequence of Cantuzumab HC-TGF3RII-PD1 fusion protein wherein the amino acid sequence for Cantuzumab heavy chain (SEQ ID NO: 29) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFpRII (immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for PD1 (immunomodulatory moiety) (SEQ ID NO: 10) is in written text font with linker between (SEQ ID No: 11) and including the amino acid sequence of Cantuzumab light chain (SEQ ID NO: 30).

[0097] Figure 63 shows the amino acid sequence of Cixutumumab HC-TGF3RII-PD1 fusion protein wherein the amino acid sequence for Cixutumumab heavy chain (SEQ ID NO: 31) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFpRII (immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for PD1 (immunomodulatory moiety) (SEQ ID NO: 10) is in written text font with linker between (SEQ ID No: 11) and including the amino acid sequence of Cixutumumab light chain (SEQ ID NO: 32).

2017201572 07 Mar 2017 [0098] Figure 64 shows the amino acid sequence of Clivatuzumab HC-TGF3RII-PD1 fusion protein wherein the amino acid sequence for Clivatuzumab heavy chain (SEQ ID NO: 33) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFpRII (immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for PD1 (immunomodulatory moiety) (SEQ ID NO: 10) is in written text font with linker between (SEQ ID No: 11) and including the amino acid sequence of Clivatuzumab light chain (SEQ ID NO: 34).

[0099] Figure 65 shows the amino acid sequence of Pritumumab HC-TGF3RII-PD1 fusion protein wherein the amino acid sequence for Pritumumab heavy chain (SEQ ID NO: 35) is attached to a linker (SEQ ID NO: 3) shown in italics and the sequence for TGFpRII (immunomodulatory moiety) (SEQ ID NO: 4) is identified in bold letters and the amino acid sequence for PD1 (immunomodulatory moiety) (SEQ ID NO: 10) is in written text font with linker between (SEQ ID No: 11) and including the amino acid sequence of Pritumumab light chain (SEQ ID NO: 36).

DETAILED DESCRIPTION OF THE INVENTION [00100] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

[00101] Definitions [00102] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the

2017201572 07 Mar 2017 description of the invention and the appended claims, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. The following terms have the meanings given:

[00103] The term “polynucleotide” as used herein means a sequence of nucleotides connected by phosphodiester linkages. Polynucleotides are presented herein in the direction from the 5’ to the 3’ direction. A polynucleotide of the present invention can be a deoxyribonucleic acid (DNA) molecule or ribonucleic acid (RNA) molecule. Where a polynucleotide is a DNA molecule, that molecule can be a gene or a cDNA molecule. Nucleotide bases are indicated herein by a single letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) and uracil (U). A polynucleotide of the present invention can be prepared using standard techniques well known to one of skill in the art.

[00104] The term, optimized as used herein means that a nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the production cell or organism, generally a eukaryotic cell, for example, a cell of Pichia, a cell of Trichoderma, a Chinese Hamster Ovary cell (CHO) or a human cell. The optimized nucleotide sequence is engineered to retain completely or as much as possible the amino acid sequence originally encoded by the starting nucleotide sequence, which is also known as the parental sequence. The optimized sequences herein have been engineered to have codons that are preferred in CHO mammalian cells; however optimized expression of these sequences in other eukaryotic cells is also envisioned herein. The amino acid sequences encoded by optimized nucleotide sequences are also referred to as optimized.The term expression as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

[00105] The term “transfection” of a cell as used herein means that genetic material is introduced into a cell for the purpose of genetically modifying the cell. Transfection can be accomplished by a variety of means known in the art, such as transduction or electroporation.

[00106] The term cancer as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical

2017201572 07 Mar 2017 cancer, skin cancer, ocular cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.

[00107] The term “transgene” is used in a broad sense to mean any heterologous nucleotide sequence incorporated in a vector for expression in a target cell and associated expression control sequences, such as promoters. It is appreciated by those of skill in the art that expression control sequences will be selected based on ability to promote expression of the transgene in the target cell. An example of a transgene is a nucleic acid encoding a chimeric fusion protein of the present invention.

[00108] The term expression vector as used herein means a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well. The term also includes a recombinant plasmid or virus that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo. Preferably the host cell is a transient cell line or a stable cell line and more preferably a mammalian host cell and selected from the group consisting of HEK293, CHO and NSO.

[00109] The tern subject, as used herein means a human or vertebrate animal including a dog, cat, horse, cow, pig, sheep, goat, chicken, monkey, rat, and mouse.

[00110] The term therapeutically effective amount as used herein means the amount of the subject compound that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.

[00111] The term pharmaceutically acceptable as used herein means the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

2017201572 07 Mar 2017 [00112] The term “recombinant as used herein means a genetic entity distinct from that generally found in nature. As applied to a polynucleotide or gene, this means that the polynucleotide is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in the production of a construct that is distinct from a polynucleotide found in nature.

[00113] The term substantial identity or substantial similarity, as used herein when referring to a nucleic acid or fragment thereof, indicates that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95 to 99% of the sequence.

[00114] The term “peptide,” “polypeptide” and “protein” are used interchangeably to denote a sequence polymer of at least two amino acids covalently linked by an amide bond.

[00115] The term “homologous” as used herein and relating to peptides refers to amino acid sequence similarity between two peptides. When an amino acid position in both of the peptides is occupied by identical amino acids, they are homologous at that position. Thus by “substantially homologous” means an amino acid sequence that is largely, but not entirely, homologous, and which retains most or all of the activity as the sequence to which it is homologous. As used herein, “substantially homologous” as used herein means that a sequence is at least 50% identical, and preferably at least 75% and more preferably 95% homology to the reference peptide. Additional peptide sequence modification are included, such as minor variations, deletions, substitutions or derivitizations of the amino acid sequence of the sequences disclosed herein, so long as the peptide has substantially the same activity or function as the unmodified peptides. Notably, a modified peptide will retain activity or function associated with the unmodified peptide, the modified peptide will generally have an amino acid sequence “substantially homologous” with the amino acid sequence of the unmodified sequence.

[00116] The term administering as used herein is defined as the actual physical introduction of the composition into or onto (as appropriate) the host subject. Any and all methods of introducing the composition into the subject are contemplated according to the present invention; the method is not dependent on any particular means of introduction and is not to be so construed. Means of introduction are well-known to those skilled in the art, and

2017201572 07 Mar 2017 preferably, the composition is administered subcutaneously or intratumorally. One skilled in the art will recognize that, although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. Local or systemic delivery can be accomplished by administration comprising application or instillation of the immunovaccines into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, intraportal, intrahepatic, peritoneal, subcutaneous, or intradermal administration. In the event that the tumor is in the central nervous system, the composition must be administered intratumorally because there is no priming of the immune system in the central nervous system.

[00117] Although chemotherapeutic agents can induce immunogenic tumor cell death and facilitate cross-presentation of antigens by dendritic ceils, tumors create a tolerogenic environment that allows them to suppress the activation of innate and adaptive immune responses and evade immunologic attack by immune effector cells. The present invention provides strategies to counteract tumor-induced immune tolerance in the tumor microenvironment and can enhance the antitumor efficacy of chemotherapy by activating and leveraging T cell-mediated adaptive antitumor immunity against disseminated cancer cells.

[00118] The present invention is based on the discovery that targeted immunomodulatory antibodies or fusion proteins of the present invention can counteract or reverse immune tolerance of cancer cells. Cancer cells are able to escape elimination by chemotherapeutic agents or tumor-targeted antibodies via specific immunosuppressive mechanisms in the tumor microenvironment and such ability of cancer cells is recognized as immune tolerance. By counteracting tumor-induced immune tolerance, the present invention provides effective compositions and methods for cancer treatment, optional in combination with another existing cancer treatment.

[00119] The present invention provides compositions and methods for producing fusion proteins that counteract immune tolerance in the tumor microenvironment and promote T cell-mediated adaptive antitumor immunity for maintenance of durable long-term protection against recurrent or disseminated cancers. These fusion proteins are designed to facilitate effective long term T cell-mediated immune responses against tumor cells by at least one of the following:

2017201572 07 Mar 2017

a. promoting death of tumor cells via enhancement of antibody-dependent cellular cytotoxicity (ADCC); and

b. increasing activation and proliferation of antitumor CD8+ T cells by negating immune suppression mediated by regulatory T cells and myeloid suppressor cells. These antitumor immune responses may be activated in tandem with the sensitization of tumor cells to immune effector-mediated cytotoxicity, thereby establishing a positive feedback loop that augments tumor cytoreduction and reinforces adaptive antitumor immunity.

[00120] In addition, the fusion proteins of the present invention are distinguished from and superior to existing therapeutic, molecules in at least one of the following aspects: (i) To counteract immune tolerance in the tumor microenvironment and promote T cell-mediated adaptive antitumor immunity for maintenance of long-term protection against recurrent or disseminated cancers (for prevention or treatment of diverse cancers); (ii) To produce immune cell compositions for adoptive cellular therapy of diverse cancers; and (iii) To serve as immune adjuvants or vaccines for prophylaxis of diverse cancers or infectious diseases.

[00121] The targeted immunostimulatory antibodies and/or fusion proteins of the invention provide the ability to disrupt immunosuppressive networks in the tumor microenvironment. Tumors employ a wide array of regulatory mechanisms to avoid or suppress the immune response. Cancer cells actively promote immune tolerance in the tumor microenvironment via the expression of cytokines and molecules that inhibit the differentiation and maturation of antigen-presenting dendritic cells (DC). The immunosuppressive cytokines and ligands produced by tumor cells include the following: (i) Transforming growth factor-beta (TGF-β); (ii) Programmed death- 1 ligand 1 (PD-LI ; B7Hl); (iii) Vascular endothelial growth factor (VEGF); and (iv) Interleukin-10 (IL-10).

[00122] In addition to blocking dendritic cell (DC) maturation, these molecules promote the development of specialized subsets of immunosuppressive CD4⁺ T cells (regulatory T cells; Treg cells) and myeloid-derived suppressor cells (MDSC). Tregs are a minority sub-population of CD4⁺ T cells that constitutively express CD25 [the interleukin-2 (IL-2) receptor cc-chain] and the forkhead box P3 (FOXP3) transcription factor. Tregs (CD4+CD25+FoxP3+ cells) maintain immune tolerance by restraining the activation, proliferation, and effector functions of a wide range of immune cells, including CD4 and

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CDS T cells, natural killer (NK) and NKT cells, B cells and antigen presenting cells (APCs) in vitro and in vivo.

[00123] The accumulation of Treg cells in the tumor microenvironment reinforces tumor immune tolerance and facilitates tumor progression and metastases. The increased expression of immunosuppressive cytokines (TGF-β; PD-L1 ) and tumor-infdtrating Tregs is correlated with a reduction of survival of patients with diverse types of cancers. The fusion proteins of the present invention inhibit key immunosuppressive molecules expressed by the targeted tumor cell or tumor-infdtrating Treg cells and myeloid suppressor cells (DCs or MDSC). As such, they provide the targeted ability to inhibit the development or function of Tregs within the tumor microenvironment.

[00124] The present invention provides a method of preventing or treating a neoplastic disease. The method includes administration to a subject in need thereof one or more fusion proteins of the present invention in combination with another anticancer therapy, wherein the anticancer therapy is a chemotherapeutic molecule, antibody, small molecule kinase inhibitor, hormonal agent, cytotoxic agent, targeted therapeutic agent, anti-angiogenic agent, ionizing radiation, ultraviolet radiation, cryoablation, thermal ablation, or radiofrequency ablation.

[00125] As used herein, the term antibody includes natural or artificial mono- or polyvalent antibodies including, but not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments. F(ab) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-id) antibodies (including, e.g., anti-id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The antibody may be from any animal origin including birds and mammals. In one aspect, the antibody is, or derived from, a human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken. Further, such antibody may be a humanized version of an antibody. The antibody may be monospecific, bispecific, trispecific, or of greater multispecificity. The antibody herein specifically include a chimeric antibody in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another

2017201572 07 Mar 2017 species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[00126] Examples of antibodies which can be incorporated into compositions and methods disclosed herein include, but are not limited, to antibodies such as trastuzumab (antiHER2/neu antibody); Pertuzumab (anti-HER2 mAh); cetuximab (chimeric monoclonal antibody to epidermal growth factor receptor EGFR): panitumumab (anti-EGFR antibody); nimotuzumab (anti-EGFR antibody); Zalutumumab (anti-EGFR mAh); Necitumumab (antiEGFR mAh); MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-447 (humanized anti-EGF receptor bispecific antibody); Rituximab (chimeric murine/human anti-CD20 mAh); Obinutuzumab (anti-CD20 mAh); Ofatumumab (anti-CD20 mAh); Tositumumab-1131 (anti-CD20 mAh); ibritumomab tiuxetan (anti-CD20 mAh); Bevacizumab (anti-VEGF mAh); Ramucirumab (anti-VEGFR2 mAh); Ranibizumab (anti-VEGF mAh); Aflibercept (extracellular domains of YEGFR1 and VEGFR2 fused to IgGl Fc): AMG386 (angiopoietin-1 and -2 binding peptide fused to IgGl Fc); Dalotuzumab (anti-IGF-lR mAh): Gemtuzumab ozogamicin (anti-CD33 mAb); Alemtuzumab (anti-Campath-l/CD52 mAb); Brentuximab vedotin (anti-CD30 mAb); Catumaxomab (bispecific mAb that targets epithelial cell adhesion molecule and CD3); Naptumomab (anti-5T4 mAb); Girentuximab (anti-Carbonic anhydrase ix): or Farletuzumab (anti-folate receptor). Other examples include antibodies such as Panorex™ (17-1 A) (murine monoclonal antibody); Panorex (@ (17-1 A) (chimeric murine monoclonal antibody); BEC2 (ami-idiotypic mAb, mimics the GD epitope) (with BCG): Oncolym (Lym-1 monoclonal antibody); SMART Μ 1 95 Ab, humanized 13' 1 LYM-1 (Oncolym), Ovarex (B43.13, antiidiotypic mouse mAb); 3622W94 mAb that binds to EGP40 ( 17- 1 A) pancarcinoma antigen on adenocarcinomas; Zenapax (SMART Anti-Tac (IL-2 receptor); SMART Ml 95 Ab, humanized Ab, humanized); NovoMAb-G2 (pancarcinoma specific Ab): TNT (chimeric mAb to histone antigens); TNT (chimeric mAb to histone antigens); GJiomab-H (Monoclonals— Humanized Abs); GN1-250 Mab; EMD-72000 (chimeric-EGF antagonist); LymphoCide (humanized IL.L.2 antibody); and MDX-260 bispecific, targets GD-2, ANA Ab, SMART IDiO Ab, SMART ABL 364 Ab or ImmuRAIT-CEA.

[00127] Various methods have been employed to produce antibodies. Hybridoma technology, which refers to a cloned cell line that produces a single type of antibody, uses the cells of various species, including mice (murine), hamsters, rats, and humans. Another

2017201572 07 Mar 2017 method to prepare an antibody uses genetic engineering including recombinant DNA techniques. For example, antibodies made from these techniques include, among others, chimeric antibodies and humanized antibodies. A chimeric antibody combines DNA encoding regions from more than one type of species. For example, a chimeric antibody may derive the variable region from a mouse and the constant region from a human. A humanized antibody comes predominantly from a human, even though it contains nonhuman portions. Like a chimeric antibody, a humanized antibody may contain a completely human constant region. But unlike a chimeric antibody, the variable region may be partially derived from a human. The nonhuman, synthetic portions of a humanized antibody often come from CDRs in murine antibodies. In any event, these regions are crucial to allow the antibody to recognize and bind to a specific antigen.

[00128] In one embodiment, a hybridoma can produce a targeted fusion protein comprising a targeting moiety and an immunomodulatory moiety. In one embodiment, a targeting moiety comprising an antibody, antibody fragment, or polypeptide is linked or fused to an immunomodulatory moiety consisting of a polypeptide, with a linker or without a linker. The linker can be an amino acid linker. In one embodiment, a linker is (GGGGS)n wherein n is 1, 2, 3, 4, 5, 6, 7, or 8. For example, GGGGSGGGGSGGGGS (SEQ ID NO: 3). In another embodiment, a linker is EPKSCDK (SEQ ID NO: 11). In various aspects, the length of the linker may be modified to optimize binding of the target moiety or the function of the immunomodulatory moiety. In various aspects, the immunomodulatory moiety is a polypeptide that is fused to the C-terminus of the Fc region of the heavy chain of a targeting antibody or Fc-containing fusion protein. In another aspect, the immunomodulatory moiety is a polypeptide that is fused to the C-terminus of the light chain of a targeting antibody.

[00129] An antibody fragment can include a portion of an intact, antibody, e.g. including the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; Fc fragments or Fc-fusion products; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragment(s). An intact antibody is one which includes an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CHI, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof tor any other modified Fc (e.g. glycosylation or other engineered Fc).

2017201572 07 Mar 2017 [00130] The fusion proteins of the present invention may be synthesized by conventional techniques known in the art, for example, by chemical synthesis such as solid phase peptide synthesis. Such methods are known to those skilled in the art. In general, these methods employ either solid or solution phase synthesis methods, well known in the art. Specifically, the methods comprise the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected, under conditions suitable for forming the amide linkage. The protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups and any solid support are removed either sequentially or concurrently to afford the final polypeptide. By simple modification of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under condition that do not racemize chiral centers) a protected tripeptide with a properly protected dipeptide to form, after deprotection, a pentapeptide.

[00131] Typical protecting groups include t-butyloxycarbonyl (Boc), 9fluorenylmethoxycarbonyl (Fmoc), benxyloxycarbonyl (Cbz), p-toluenesulfonyl (Tos); 2,4dinitrophenyl, benzyl (Bzl), biphenylisopropyloxy-carboxycarbonyl, cyclohexyl, isopropyl, acetyl, o-nitrophenylsulfonyl, and the like. Of these, Boc and Fmoc are preferred.

[00132] Typical solid supports are generally cross-linked polymeric materials. These include divinylbenzene cross-linked styrene-based polymers, for example, divinylbenzenehy dr oxy methyl styrene copolymers, divinylbenzene-chloromethylstyrene copolymers, and divinylbenzene-benzhydrylaminopolystyrene copolymers. The divinylbenzenebenzhydrylaminopolystyrene copolymers, as illustrated herein using p-methylbenzhydrylamine resin, offers the advantage of directly introducing a terminal amide functional group into the peptide chain, which function is retained by the chain when the chain is cleaved from the support.

2017201572 07 Mar 2017 [00133] In one method, the polypeptides are prepared by conventional solid phase chemical synthesis on, for example, an Applied Biosystems, Inc. (ABI) 430A peptide synthesizer using a resin that permits the synthesis of the amide peptide form and using t-Boc amino acid derivatives (Peninsula Laboratories, Inc.) with standard solvents and reagents. Polypeptides containing either L- or D-amino acids may be synthesized in this manner. Polypeptide composition is confirmed by quantitative amino acid analysis and the specific sequence of each peptide may be determined by sequence analysis.

[00134] Preferably, the polypeptides can be produced by recombinant DNA techniques by synthesizing DNA encoding the desired polypeptide. Once coding sequences for the desired polypeptides have been synthesized or isolated, they can be cloned into any suitable vector for expression. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. The gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as control elements), so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the host cell transformed by a vector containing this expression construction. The coding sequence may or may not contain a signal peptide or leader sequence. Heterologous leader sequences can be added to the coding sequence that causes the secretion of the expressed polypeptide from the host organism. Other regulatory sequences may also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell. Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.

[00135] The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.

[00136] The expression vector may then used to transform an appropriate host cell. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to,

2017201572 07 Mar 2017

Chinese hamster ovary (CHO) cells, HeLa cells, HEK293, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), MadinDarby bovine kidney (MDBK) cells, NOS cells derived from carcinoma cells, such as sarcoma, as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni. The proteins may also be expressed in Trypanosomes.

[00137] Depending on the expression system and host selected, the proteins of the present invention are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The protein is then isolated from the host cells and purified. If the expression system secretes the protein into growth media, the protein can be purified directly from the media. If the protein is not secreted, it is isolated from cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art. Once purified, the amino acid sequences of the proteins can be determined, i.e., by repetitive cycles of Edman degradation, followed by amino acid analysis by HPLC. Other methods of amino acid sequencing are also known in the art.

[00138] Once synthesized or otherwise produced, the inhibitory activity of a candidate polypeptide can be tested by assessing the ability of the candidate to inhibit the lipopolysaccharide-induced nuclear translocation of NF-.kappa.B by, for example, using murine endothelial cells.

[00139] The fusion proteins of the present invention can be formulated into therapeutic compositions in a variety of dosage forms such as, but not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules or microvesicles, liposomes, and injectable or infusible solutions. The preferred form depends upon the mode of administration and the particular cancer type targeted. The compositions also preferably include pharmaceutically acceptable vehicles, carriers or adjuvants, well known in the art,

2017201572 07 Mar 2017 such as human serum albumin, ion exchangers, alumina, lecithin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, and salts or electrolytes such as protamine sulfate. Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Actual methods of preparing such compositions are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18th edition, 1990.

[00140] The above compositions can be administered using conventional modes of delivery including, but not limited to, intravenous, intraperitoneal, oral, intralymphatic, or subcutaneous administration. Local administration to a tumor in question, or to a site of inflammation, e.g., direct injection into an arthritic joint, will also find use with the present invention.

[00141] Therapeutically effective doses will be easily determined by one of skill in the art and will depend on the severity and course of the disease, the patient's health and response to treatment, and the judgment of the treating physician.

[00142] Experimental [00143] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[00144] Example 1 [00145] The Fusion proteins comprising of IgG heavy chain linked to immunomodulator (either suppressor or activator) ligands were expressed by codon optimized genes for the expression of CHO cells. The codon optimized nucleotide sequences defined by SEQ ID NOs: 12 to 28 were expressed in (CHO) cells and the expressed chimeric/fusion proteins are shown in Table 1

Fusion protein Details

2017201572 07 Mar 2017

Anti-HER2/neu heavy chain + TGF3-RII ECD and Anti-HER2/neu light chain

Anti-EGFRl heavy chain + TGF3-RII ECD and Anti- EGFR1 light chain

Anti-CTLA4 heavy chain + TGF3-RII ECD and Anti-CTLA4 light chain

Anti-CTLA4 heavy chain + PD1 ectodomain and Anti-CTLA4 light chain

Anti-HER2/neu heavy chain + 4-1BBL and Anti-HER2/neu light chain

Anti-EGFRl heavy chain + 4-1BBL and Anti- EGFR1 light chain

Anti-CTLA4 heavy chain + 4-1BBL and Anti-CTLA4 light chain

PD1 ectodomain-Fc-4-lBBL

TGFpRII ECD-Fc-4-lBBL

Anti-EGFRl heavy chain + PD1 ectodomain and Anti- EGFR1 light chain

Anti-CD20 heavy chain + 4-1 BBL and Anti-CD20 light chain

Anti-HER2/neu heavy chain + PD1 ectodomain and Anti-HER2/neu light chain

Anti-IL6Rheavy chain + PD1 ectodomain and Anti-IL6R light chain

Anti-IL6Rheavy chain + TGF3-RII ECD and Anti-IL6R light chain

Anti-4-IBB heavy chain + PD1 ectodomain and Anti-4-IBB light chain

[00146] The expressed protein were characterized by using SDS PAGE and the expressed fusion proteins Anit-HER2/neu-TGF3RII and Anti-EGFRl- TGFpRII were purified from culture supernatants using ProteinA column and the results are shown in Figure 22. Notably, Anti-EGFRl-TGFpRII light chain mass is higher and it may be because of the presence of two glycosylation sites on the variable regions light and heavy chain. Both the Anti-HER2/neu-TGF3RII & Anti-EGFRl-TGFpRII heavy chains mass are higher because of the TGFpRIE Also Anti-HER2/neu-TGF3RII heavy chain has four N-glycosylation sites while Anti-EGFRl-TGFpRII has five N-glycosylation sites.

[00147] Example 2 [00148] Protein A/SEC chromatography. The Anti-HER2/neu-TGF3RII and AntiEGFRl -TGFpRII samples were analyzed by ProteinA/SEC chromatography and the results are shown in Figure 23. Figure 23 A shows a sharp peak of elution of Bmab200(Herceptin)

2017201572 07 Mar 2017 vs a broader elution peak is believed to be a measure of heterogeneity due to presence of glycosylation as there are three additional N-glycosylation sites that are present in the TGFpRII region. Notably storage at -80C did not causing aggregation. The shift in the position or appearance of the peak early in SEC column indicates that the increase in the molecular weight is because of the fusion partner. This once again confirms that the full length molecule is being expressed. Figure 23 B shows a sharp peak of elution of Bmab200(Herceptin) vs a broader elution peak which is believed to be a measure of heterogeneity due to presence of glycosylation sites as there are three additional Nglycosylation sites are present in the TGFpRII region. Again, storage at -80C did not causing aggregation. The shift in the position or appearance of the peak early in SEC column indicates that the increase in the molecular weight is because of the fusion partner. This once again confirms that the full length molecule is being expressed.

[00149] Example 3 [00150] Functional assays for the Fusion proteins. ELISA experiment was carried out to check the binding ability of Anti-HER2/neu-TGF3RII and Anti-EGFRl-TGFpRII to TGFp. Figure 24 A shows that Anti-HER2/neu-TGFpRII and Anti-EGFRl-TGFpRII molecules bind to the TGF3 indicating that the fusion protein is functional. Figure 24 B shows that Anti-HER2-TGF3RII inhibits the proliferation of BT474 cell line similar to the Bmab200 (Herceptin). Figure 25 shows that Anti-EGFRl-TGFpRII-inhibits the proliferation of A431 cell line similar to the Cetuximab.

[00151] Example 4 [00152] Antibody dependent cellular cytotoxicity ADCC activity for Anti-HER2/neuTGFpRII fusion protein was conducted to determine that the protein binds to the target receptors on the cells. The results are shown in Figure 26 wherein the activity is determined in BT474 cells and it is evident that ADCC activity (%lysis of cells) of Anti-HER2-TGF3RII on BT474 cells is similar to that of Bmab200(Herceptin). Figure 27 shows ADCC activity of Anti-EGFRl-TGFpRII on A431 cells wherein the ADCC activities are similar to that of Cetuximab. Figure 28 shows the ADCC activity of ADCC activity of Anti-EGFRl-4-IBB in comparison with Anti-EGFRl-TGFpRII and cetuximab.

2017201572 07 Mar 2017 [00153] Example 5 [00154] Binding Activity of the expressed proteins. The aim of this assay is to test the functionality of the fusion proteins to bind to the target receptors on the cells in a dose dependent manner. Figure 29 A shows that the binding activity of Anti-CTLA4-TGF3RII to TGFpi is comparable to Anti-EGFR1-TGF3RII and B shows that the binding activity of Anti-CTLA4-TGF3RII to CTLA4. Figure 30 A shows the binding activity of Anti-CTLA4TGFpRII to determine the level of PDl-Fc binding and B shows the binding activity of AntiEGRF1-4-1BB to determine the binding of 4-1BBL. Figure 31 A shows the binding activity of Anti-EGFRl-4-lBB to EGFR and B shows the binding activity of PDl-Fc-4-lBB to find out PDLl-Fc. Figure 32 shows the binding activity of Anti-EGFRl-PDl to EGFR and PD1.

[00155] Example 6 [00156] Confirmation of primary structure of molecule. As shown in Figure 33, the expressed proteins are evaluated to determine the molecular weight and the presence of glycosylation. The samples were analyzed by reducing and non-reducing SDS PAGE. The heavy and light chains of the antibody are separated by reduction alkylation so that the reduced structures can be evaluated. Tryptic digestion of the fusion proteins provides for the identification of the primary sequence. MS/MS analysis of the proteins is performed.

[00157] Mass Spectrometry Analysis of Anti-HER2/neu-TGF3RII and Anti-EGFRlTGFpRII. The fusion protein shown in Figure 1 was expressed and tested. Figure 34 A shows the mass spectrum Mass Spectrum of light chain (LC )(Reduced) of Anti-HER2/neuTGFpRII ECD fusion and B shows Deconvoluted Mass Spectrum of LC (Reduced) of AntiHER2/neu-TGF3RII ECD fusion. Figure 35 shows the Mass Spectrum of heavy chain (HC) (Reduced) of Anti-HER2/neu-TGF3RII ECD fusion.

[00158] The fusion protein shown in Figure 2 was expressed and tested. Figure 36 A shows the Mass Spectrum of LC (Reduced) of Anti-EGFRl-TGFpRII ECD and B shows the Deconvoluted Mass Spectrum of LC (Reduced) of Anti-EGFR1-TGF3RII ECD. Figure 37 shows the Mass Spectrum of HC (Reduced) of Anti-EGFRl-TGFpRII ECD.

[00159] Example 7

2017201572 07 Mar 2017 [00160] The fusion proteins having amino acid sequences as described in Figures 1 and 2 were inspected using UV chromatography and providing chromatograms resulting from the chromatographic separation of the tryptic digest of the fusion proteins and tested with UV 218-222 nm wavelength. Total Ion Current (TIC) corresponding to UV trace was also evaluated. Figure 38 A shows the UV Chromatogram of Tryptic Peptides of Anti-HER2/neuTGFpRII ECD fusion protein and B shows the Total Ion Chromatogram (TIC) of Tryptic Peptides of Anti-HER2/neu-TGF3RII ECD fusion protein. Figures 39, 40 and 41 provide lists of expected/observed tryptic peptide of the light chain, heavy chain and linked motif of the Anti-HER2/neu-TGF3RII ECD fusion protein, respectively. Notably, all the expected peptides of the molecules were identified including the light and heavy chain peptides and the peptides of the linked motif (TGF βΙΗΙ).

[00161] Figure 42 A shows the UV Chromatogram of Tryptic Peptides of AntiEGFRl-TGE^RII ECD fusion protein and B shows the Total Ion Chromatogram (TIC) of Tryptic Peptides of Anti-EGFRl-TGE^RII ECD fusion protein. Figures 43, 44, and 45 provide lists of expected/observed tryptic peptide of the light chain, heavy chain and linked motif of the Anti-EGFRl-TGE^RII ECD fusion protein, respectively. Again all the expected peptides of the molecules were identified including the light and heavy chain peptides and the peptides of the linked motif (TGF βΚΙΙ).

[00162] Example 8 [00163] The host cell line used for the expression of recombinant fusion protein expression is CHO cells or the derivative of the CHO cells. The CHO cells referred here is either freedom CHO-S cells; CHO-S Cells are CHO-derived cells adapted to high density, serum-free suspension culture in chemically-defined medium that are capable of producing high levels of secreted, recombinant protein or CHO K1 cells; having the same as ATCC No. CCL-61. It is basically an adherent cell line. The vectors used for stable cell line:

[00164] The Freedom pCHO 1.0 vector, designed by ProBioGen AG, to express one or two genes of interest downstream of the vector's two different hybrid CMV promoters. This vector contains the dihydrofolate reductase (DHFR) selection marker and a puromycin resistance gene, allowing selection using MTX and Puromycin simultaneously.

2017201572 07 Mar 2017 [00165] The light chain or the light chain fusion protein coding nucleic acid sequences are cloned into the restriction enzyme sites Avril and BstZ17 under the control of EF2/CMV promoter. The heavy chain or the heavy chain fusion protein coding nucleic acid sequences are cloned, in restriction enzyme sites EcoRV and Pad under the control of CMV/EF1 promoter.

[00166] The construct(s) are transfected into Freedom CHO-S cells/CHOKl cells. The high producer single, clonal cell strain is selected for producing the recombinant fusion protein. Prepare the MCB and characterize for cell viability, productivity, stability and other parameters. The cells are used for culturing followed by purification.

[00167] Example 9 [00168] The cell culture is performed in feed-batch mode. In the cell culture, the mammalian host cells used is Chinese Hamster Ovary (CHO) cells and culture medium are supplied initially. The CHO cells are genetically engineered to produce the Antibody-peptide fusion protein. The zinc sulphate hepta hydrate salt is added in the medium at a concentration of 0.4 mM. In contrast, there is no addition of any zinc salt in the control medium. The production fermentation run starts with an initial cell count of 0.3-0.45xl0⁶cells/ml at 37 ± 1°C, the first 3-4 days are dedicated to grow the cells in batch phase. Next step involves lowering the temperature to 31±1 °C and continuing the run till 7th day. Lactate reduces by almost 10-40% throughout the run. The produced fusion protein is then collected from the media using the technique of affinity chromatography.

[00169] Example 10 [00170] The cell culture is performed in a feed-batch mode is employed. In the cell cultures the mammalian host cells and culture medium which is Hyclone CDM4Mab are supplied initially. The salts (zinc) is also added in the medium (0.3mM). The production fermentation run starts with an initial cell count of 0.3-0.45xl0⁶ cells/nil at 37 ± 1° C, the first 3-4 days are dedicated to growing the cells in batch phase. Next step involves lowering the temperature to 31+1-1 °C and continuing the run till 7th day.

2017201572 07 Mar 2017 [00171] Example 11 [00172] Purification of antibody-peptide fusion immunostimulatory molecules using protein A column. Supernatant culture secreted from recombinant CHO cell line containing the fusion monoclonal antibodies is tested for titer and endotoxins under sterile conditions. The supernatant is subjected to affinity chromatography using Mab Select Xtra Protein A affinity resin, washed and equilibrated with binding buffer. The pH of the supernatant is adjusted using 0.5M phosphate to the same pH as the column; the supernatant is allowed to bind to the column/ pass through the column at the flow rate of 0.5 ml/minute to achieve the maximum binding. All the Antibody-proteins fusion molecules bind through the Fc region while impurities are eliminated as flow through.. The column is washed with equilibration buffer and the bound fusion molecules are eluted using 0.1 M glycine at pH 3.0. The pH of the eluted proteins is adjusted to neutral pH or the stable formulation pH and the purified protein are stored at -20°C or at 2-8°C.

[00173] Example 12 [00174] Differentiating Trastuzumab from Trastuzumab-TGF βΙΗΙ receptor fusion molecule [00175] A breast cancer tumor overexpressing the ErbB2 receptor will either by constitutive activation or heterodimerization with other members of the ErbB family of receptors lead to tumor progression. This will involve the binding of growth factors associated with the ErbB signaling pathway. In addition to this, the tumor creates a milieu wherein the immune system is suppressed by activating TGF β and specific cytokines involved in the subdued immune response. A novel molecule is generated wherein Trastuzumab (anti ErbB2) is fused with the TGF βΙΗΙ receptor as a fusion protein. While it is hypothesized that Trastuzumab will act as a targeted molecule homing into the ErbB2 overexpressing breast cancer cells, the ΤΟΡβΙΗΙ receptor will sequester ΤΟΡβ leading to immune activation. The experiment will utilize the growth of Herceptin resistant ErbB2 expressing cell lines (selected by growing BT474 cells in the presence of Herceptin) in the presence of ΤϋΕβ, cytotoxic CD8 positive cells and NK cells. While Trastuzumab will be ineffective in inducing cytotoxicity Trastuzumab ΤΟΡβΙΗΙ receptor fusion molecule will sequester the ΤΟΡβ thereby preventing the inhibition of cytotoxic CD8 and NK cells. This

2017201572 07 Mar 2017 will lead to enhanced cytotoxicity observed in Trastuzumab -TGFpRII receptor fusion treated cells over cells treated with Trastuzumab alone. The readout for the experiment will use Alamar Blue a resazurin dye which will get activated directly proportional to live cells present. Another method could be to measure cytotoxicity by using cytotox gio which measures protease release which directly corresponds to proportional dead cells. Yet another method could be the use of the flow cytometer directly measuring apoptotic and necrotic cell population by using Annexin V and propidium iodide. Results from these multiple experiments will elucidate understanding of the activity of the conjugate molecule as compared to Trastuzumab alone.

[00176] Although the invention has been described with reference to the above example, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

2017201572 29 May 2018

Claims

Claims

That which is claimed is:

1. A chimeric fusion protein comprising a targeting moiety to target a cancer cell and an immunomodulating moiety that counteracts immune tolerance, wherein the targeting moiety and the immunomodulating moiety are linked by an amino acid spacer selected from SEQ ID NO: 3 or SEQ ID NO: 11, wherein the immunomodulating moiety is TGF-/3RII SEQ ID NO: 4, wherein the targeting moiety is anti-CTLA4 consisting of heavy chain SEQ ED NO: 7 and light chain SEQ ID NO: 8; wherein SEQ ED NO: 4 is attached via the amino acid spacer to the C-terminus of SEQ ID NO 7 or SEQ ID NO: 8.
2. A method of treating a neoplastic disease, the method comprising the administration to a subject in need thereof a chimeric fusion protein according to claim 1.
3. A process of preparing the chimeric fusion protein according to claim 1, comprising: transfecting a host cell with polynucleotide sequences that encode the chimeric fusion protein and maintaining the transformed host cell under biological conditions sufficient for expression of the chimeric fusion protein.
4. A method of preparing the chimeric fusion protein according to claim 1, the method comprising:

a) preparing a codon optimized sequence of the said chimeric fusion protein;

b) cloning the optimized sequence of said chimeric fusion protein a host cell capable of transient or continued expression;

c) growing the host cell in a media sufficiently and allowing it to express the cloned chimeric fusion protein; and

d) subjecting the expressed chimeric fusion protein to purification and optionally checking the bi-specific binding capabilities of the chimeric fusion protein to its targets.
5. The method of claim 4, wherein the immunomodulating moiety is either bound to the C-terminus of the heavy or light chain of the antibody.
6. The method of claim 4, wherein the host cell is a transient cell line or a stable cell line

3665046v1
7. The method of claim 6, wherein transient expression is done by transfecting or transforming the host with vectors carrying the chimeric fusion proteins into mammalian host cells

2017201572 29 May 2018
8. The method of claim 7, wherein the transiently expressed chimeric fusion peptides are subjected to purification and in-vitro tests to check its bi-specificity
9. The method of claim 8, wherein the in-vitro test are ELISA or NK/T-cell binding assays to validate bi-functional target binding or immune cell stimulation.
10. The method of claim 9, wherein the peptides demonstrating desired bi-specificity are selected for sub-cloning into a stable cell line for larger scale expression and purification.
11. The method of claim 10, wherein the expression levels in stable cell line are comparable to the earlier generations.
12. The chimeric fusion protein of claim 1, for use in treating a neoplastic disease.
13. The chimeric fusion protein of claim 1, when used for treating a neoplastic disease.
14. Use of the chimeric fusion protein according to claim 1 in the manufacture of a composition for treating a neoplastic disease.

3665046vl

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Anti-HER2/neu-TGFpRII fusion protein at LC constant region

Amino acid sequence of Anti-HER2/neu heavy chain:

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRGAP

GKGLEWVAR1YPTNGYTRYADSVKGRFTISADTSKNTAYLQM

NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG

ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN

HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHN

AKTKPREEQYNSTYRWSVLTVLHGDWLNGKEYKCKVSNKA

LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV

DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Amino acid sequence of Anti-HER2/neu light chain fusion protein:

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPG

KAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTiSSLQPEDFA

TYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFiFPPSDEQLKS

GTASWCLLNNFYPREAKVGWKVDNALQSGNSQESVTEGD

SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF

NRGECGGGGSGGGGSGGGGSTIPPHVQKSVNNDM1VTDNN

GAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEV

CVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE

KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

Figure 1

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Anti-EGFR1-TGFpRII fusion protein at LC constant region

2017201572 07 Mar 2017

Amino acid sequence of Anti-EGFR1 heavy chain:

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSP

GKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKM

NSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKG

PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT

SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKP

SNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

DTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKT

KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA

PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL.TCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS

RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Amino acid sequence of Anti-EGFR1 light chain fusion protein:

DiLLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGS

PRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYY

CQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGT

ASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK

DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR

GECGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGA

VKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCV

AVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKK

KPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

Figure 2

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Anti-CTLA4-TGFpRll fusion protein at LC constant region

2017201572 07 Mar 2017

Amino acid sequence of anti-CTLA4 heavy chain:

QVQLVESGGGWQPGRSLRLSCAASGFTFSSYTMHWVRQA

PGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQ

MNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKG

PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT

SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKP

SNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

DTLM1SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKT

KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA

PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS

RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Amino acid sequence of anti-CTLA4 light chain fusion protein:

EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKP

GQAPRLLIYGAFSRATGlPDRFSGSGSGTDFTLTiSRLEPEDF

AVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQL

KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS

FNRGECGGGGSGGGGSGGGGSTIPPHVQKSVNNDM1VTDN

NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQE

VCVAVWRKNDENITLETVCHDPKLPYHDF1LEDAASPKCIMK

EKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

Figure 3

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Anti-HER2/neu HC-4-1BB and LC-TGFpRII fusion protein:

Amino acid sequence of heavy chain-4-1 BB fusion protein:

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW

VARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYY

CSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG

TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW

IVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL

LGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGS4CPRWV5G

WGPLSWS^GLAGVSLrGGLSYKFVTKFLWAT^GWyvrFQLFL

1Z1ZWA<5^:£GSGSVSLAL^:KL^f^>LlZSAAGAAALALTVDL^:P^:PASSFA1iNS

AFGFQGKLLHLSAGQIZLGWLHrFAKAHHAWQLrQGATVLGLFn

VrptlPAGLPSPIZSt

Amino acid sequence of fight chain-TGFpRII fusion protein:

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI

YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQGHYTTPP

TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAK

VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVQKS

VNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICE

KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEK

KKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

Figure 4

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Anti-EGFR1 HC-4-1BB and LC-TGFpRII fusion protein:

2017201572 07 Mar 2017

Amino acid sequence of heavy chain-4-1 BB fusion protein:

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLE

WLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYY

CARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGT

AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT

VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL

GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTiSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGS4CPUMVSG

AHASPGSAAS'PHLHEQPELSFDOPA(3LLOLKQ(fMFA^LVAQNVLL

IOQPLSWYSVFQLA(^VSLrGGLSYKFVFK.FLWAKA(^VWFF^LEL

KKWAGFQSGSVSLALHLQFLIiSAA^AAALALlVVLPFASSFAKNS

AF^F^F-LLHLSAGQHLQVHLHrFAHAnFAWQLTQQATVL^LFF

VFPFIFAGUPSFKSF

Amino acid sequence of light chain-TGF£RII fusion protein:

DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKY

ASESiSGIPSRFSGSGSGTDFTLSINSVESEDiADYYCQGNNNWPTTFG

AGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQ

WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVQKSVN

NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKP

QEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKK

KPGETFFMCSCSSDECNDNilFSEEYNTSNPD

Figure 5

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Anti-CTLA4 HC-4-1BB and LC-TGFpRU fusion protein:

Amino acid sequence of heavy chain-4-1 BB fusion protein:

QVQLVESGGGWQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLE

WVTFISYDGNNKYYADSVKGRFTiSRDNSKNTLYLQMNSLRAEDTAlY

YCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA

ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTV

PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG

GPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI

EKTISKAKGQPREPGVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE

WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTGKSLSLSPGGGGGSGGGGSGGGGSACPWAVSGA nASPGSAASPKLKFGP^LSPVVPAGiLVLKQ.^FAQLVA^VLLI

OGPl^WySOP^LA^VSLrQGLSYKFOTKFLWAKAGVYYVFF^LELK

KWAGFGSGSVSLALHL^LKSAAGAAALALTVDLPrASSFAmSA

FGFQGnLLHLSAGQHLGVHLHTEAHAIlHAWQLTQ.GATVLGiFRy miFAGLPSPKSF

Amino acid sequence of light chain-TGFpRII fusion protein:

EIVLTGSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLL

IYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP

WTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPRE

AKVQWKVDNALQSGNSQESVTEQOSKDSTYSLSSTLTLSKADYEKHK

VYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVQ

KSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSI

CEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK

EKKKPGETFFMCSCSSDECNDNI1FSEEYNTSNPD

Figure 6

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Anti-HER2/neu HC-PD1 and LC-TGFpRII fusion protein:

2017201572 07 Mar 2017

Amino acid sequence of heavy chain-PD1 fusion protein:

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW

VARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYY

CSRWGGDGFYAMDYWGGGTLVTVSSASTKGPSVFPLAPSSKSTSGG

TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW

TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL

LGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTiSKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSiWFWSP mrwNi^PTrsrALLvvTr^ONArFrcsFSNrsEswLNwriiHsrsN

S^rYLC^AISLAPKAQlKFSLIZAFiHVrEmAFVFTAHFSFSPKFA

GQfQTLV

Amino acid sequence of light chain-TGFpRII fusion protein:

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI

YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP

TFGQGTKVEIKRTVAAPSVFiFPPSDEQLKSGTASWCLLNNFYPREAK

VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVQKS

VNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSiCE

KPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEK

KKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

Figure 7

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Anti-EGFR1 HC-PD1 and LC-TGFpRII fusion protein:

Amino acid sequence of heavy chain-PD1 fusion protein:

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLE

WLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAiYY

CARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGT

AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT

VPSSSLGTQTYiCNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL

GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSP^WFtDSP mpWKpprpspALLwrEGONArrrcsFsiQrsEspvLNWYRHSPSb}

Scr/LCcAISLAPKAQIKPSLPAPLPVrPPKAPWTAHPSPSPPPA

GQfQTLV

Amino acid sequence of light chain-TGFpRII fusion protein:

DiLLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKY

ASESISGI PSRFSGSGSGTDFTLSI NSVESEDIADYYCQQN N N WPTTFG

AGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQ

WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVQKSVN

NDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKP

QEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKK

KPGETFFMCSCSSDECNDNHFSEEYNTSNPD

Figure 8

2017201572 07 Mar 2017 y/os

Anti-CTLA4 HC-PD1 and LC-TGFpRII fusion protein.

Amino acid sequence of heavy chain-PD1 fusion protein:

QVQLVESGGGWQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLE

WVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIY

YCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA

ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTV

PSSSLGTQTYiCNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG

GPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI

EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE

WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGS?W?W5Pi?

mVN'PPT'FSPALLWTFGONATfTCSFSNTSTS^LNWY'R.MS'PSNQ ^TYLCQAISLAPKAQIKESLPAELIZVTEmAV/PrAHPSPSPKPA^ ororiv

Amino acid sequence of light chain-TGFpRII fusionprotein:

EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLL

IYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP

WTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPRE

AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK

VYACEVTHGGLSSPVTKSFNRGECGGGGSGGGGSGGGGSTIPPHVQ

KSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSI

CEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK

EKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

Figure 9

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Anti-HER2/neu HC-TGFpRU-4-1BB fusion protein

Amino acid sequence of heavy chain-TGFpRII-4-1BB fusion protein:

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW

VARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYY

CSRWGGDGFYAMDYWGQGTLV7VSSASTKGPSVFPLAPSSKSTSGG

TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLGSSGLYSLSSW

TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL

LGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRWSVLIVLHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVQK

SVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSIC

EKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE

KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDEPKSCDOCPUW

SWRASPGSAASPRLT&GPELSPOVPAGLLOLRQGMFAQLVAQNV

LLIOGPLSWYS'DrGiAGVSLrGGLSYKEOTlCELWAKACVYYVrr^L

ELKKWAGEGSGSVSLALHLQPLHSAAGAAALALTVDLrFASSEA'R,

NSAFGFQGHLLHiSAG^LGVHLHrEAMmAWQLrQGATVLGL bKVr'PEI'PACLPSPPSP

Amino acid sequence of light chain:

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQGKPGKAPKLLI

YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP

TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAK

VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

Figure 10

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Anti-EGFR1 HC-TGFpRll-4-1BB fusion protein

Amino acid sequence of heavy chain-TGFPRI 1-4-1 BB fusion protein:

QVQLKQSGPGLVQPSQSLSiTCTVSGFSLTNYGVHWVRQSPGKGLE

WLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYY

CARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGT

AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT

VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL

GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTfSKAKGQPREPQVYTLPPSRDELTKNGVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVQK

SVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSIC

EKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE

KKKPGETFFMCSCSSDECNDNIlFSEEYNTSNPDEPKSCDK/tCPUW

LLLVGHLSWYSVHGLAGVSLTGGLSYKEOrK.ELVVAKAGVYYVEEQL ELKKWAGEGSGSVSLA LH LQHLHSAAGAAA LA LTVOLPfASSEAH ^SAEGEQGKLLHLSA<SQHL(5VHLHrEAnA1iHAW^Lr^AlVLGL EHYTHEIHA^LHSHRSE

Amino acid sequence of light chain:

DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKY

ASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFG

AGTKLELKRTVAAPSVFIFPPSDEGLKSGTASWCLLNNFYPREAKVQ

WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGEC

Figure 11

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Anti-CTLA4 HC-TGFpRII-4-1BB fusion protein

Amino acid sequence of heavy chain-TGFpRII-4-1BB fusion protein,

QVQLVESGGGWQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLE

WVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIY

YCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA

ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLGSSGLYSLSSWTV

PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG

GPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRWSVLTVLHGDWLNGKEYKCKVSNKALPAPI

EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE

WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVQKSV

NNDM1VTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEK

PQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKK

KPGETFFMCSCSSDECNDNilFSEEYNTSNPDHPKSCDKACPVlMVSG

AHASPQSAASPKLH^PELSPWPA^LL'DLIZQGHPAQLVAQMVLL lO^LSWYSVrGLA^VSLr^LSYK^OTTaLWAKAGVWrrQL^L 'IZHVYA^^SGSVSLALHLQ'PL'RSAACAAALALTVOLDPASSFAK^S

Af^rQQKLLHLSAGQ^LiSVHLH'iTAnAKHAWQLrQ^ATVLGLFiZ

VTf^IPAQLPSmSE

Amino acid sequence of light chain:

EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLL IYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP WTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPRE AKVQWKVDNALQSGNSGESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYAC EVTHQGLSS PVTKS FN RGEC

Figure 12

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Anti-HER2/neu HC-TGFpRII-PDI fusion protein

Amino acid sequence of heavy chain-TGFpRII-PD1 fusion protein:

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW

VARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYY

CSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG

TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW

TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL

LGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVQK

SVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSIC

EKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE

KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDEFKSCZXFWTW

SPOHrWNfPTfSPALLWrEGONArfTCSFSMrSESr/LNWYRMSrf

SNQr'DKLAAFPFmSQ.PGQ.VCnFPVrQLPNGPVFHMSWnAm

FlVSGTyLCGAISLAPKAQIKESLHAFL'RX/rF'iZIZAFVprAHPSPSPH

PAGQFQTLV

Amino acid sequence of light chain:

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI

YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP

TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAK

VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

Figure 13

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Anti-EGFR1 HC-TGFpRII-PD1 fusion protein;

Amino acid sequence of heavy chain-TGFpRII-PD1 fusion protein:

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLE

WLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYY

CARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGT

AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT

VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL

GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGV

EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP

APiEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS

CSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVQK

SVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSIC

EKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKE kkkpgetffmcscssdecndniifseeyntsnpdepkscdkpgwld

SPOHVWNVPrfS'PALLWTL^ONArFrCSFSNTSLSfVLNWYNMSP bJOSGTYLC^AISLAPKAQIKESLIiAEL'RMVEKKAFVprAHPSPSPK fMGQFQTLV

Amino acid sequence of light chain:

DILLTQSPViLSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKY

ASESISGIPSRFSGSGSGTDFTLSiNSVESEDIADYYCQQNNNWPTTFG

AGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQ

WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGEC

Figure 14
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Anti-CTLA4 KC-TGFpRII-PD1 fusion protein

Amino acid sequence of heavy οΚθίη-ΤβΡβΡΙΙ-ΡΟΙ fusion protein:

QVQLVESGGGWQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLE

WVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAiY

YCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA

ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTV

PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG

GPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI

EKTISKAKGGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE

WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSTIPPHVQKSV

NNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEK

PQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKC1MKEKK

KPGETFFMCSCSSDECNDNIlFSEEYNTSNPDEPKSCDKRGWFLnSi⁵

SGTYLCGAISLAPKAQIKESLKAELRVrEKKAUWrAHPS'PS'pn'PA

GQFQTLV

Amino acid sequence of light chain:

EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLL

IYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP

WTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPRE

AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK

VYACEVTHQGLSSPVTKSFNRGEG

Figure 15
16/65

Nucleotide sequence of Anti-HER2/neu heavy chain constant region with linker:

2017201572 07 Mar 2017

1 gctagcacca agggcccctc cgtgttccct ciggccccct ccagcaagtc cacctctggc

61 ggcaccgccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc 121 tggaactctg gcgctctgac ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc 181 ggcctgtact ccctgtcctc cgtcgtgacc gtgccctcca gctctctggg cacccagacc 241 tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa ggtggaaccc 301 aagtcctgcg acaagaccca cacctgtcccccctgccctg cccctgagct cctgggaggc 361 cctagcgtgt icctgitccc cccaaagccc aaggacaccc tgatgatctc ccggaccccc 421 gaagtgacct gcgtggtggt ggacgtgtcc cacgaggacc ctgaagtgaa gttcaattgg 481 tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ccagagagga acagtacaac 541 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaaa 601 gagtacaagt gcaaggtgtc caacaaggcc ctgcctgccc ccatcgaaaa gaccatctcc 661 aaggccaagg gccagccccg cgagcctcag gtgtacaccc tgccccctag ccgggaagag 721 atgaccaaga accaggtgtc cctgacctgt ctggtcaagg gcttctaccc ctccgatatc 781 gccgtggaat gggagtccaa cggccagccc gagaacaact acaagaccac cccccctgtg 841 ctggactccg acggctcatt cttcctgtac tccaagctga ccgtggacaa gtcccggtgg 901 cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 961 cagaagtccc tgtccctgag cccaggcaaa ggcggaggcg gatctggcgg cggaggatct

....1021 ggtggcggatcc

Nucleotide sequence of TGFpRII ECD:

1 ggatccacca tccccccaca cgtgcagaaa tccgtgaaca acgacatgat cgtgaccgac

61 aacaacggcg ctgtgaagtt cccccagctg tgcaagttct gcgacgtgcg gttctctacc 121 tgcgacaacc agaaatcctg catgtccaac tgctccatca cctccatctg cgagaagccc 181 caggaagtgt gcgtcgccgt ctggcggaag aacgacgaga acatcaccct ggaaaccgtg 241 tgccacgacc ccaagctgcc ctaccacgac ttcatcctgg aagatgccgc ctcccccaag 301 tgcatcatga aggaaaagaa gaagcccggc gagactttct tcatgtgcag ctgctcctcc 361 gacgagtgca acgacaacat catcttctcc gaagagtaca acacctccaa ccccgactga 421 agctt

Figure 16
17/65

2017201572 07 Mar 2017

Nucleotide sequence of Anti-HER2/neu heavy chain variable region

1 gcggccgcca tgaacttcgg cctgcggctg atcttcctgg tgctgaccct gaagggcgtg 51 cagtgcgagg tgcagctggt ggaatccggc ggaggcctgg tccagcctgg cggatctctg 121 agactgtcct gcgccgcctc cggcfctcaac atcaaggaca cctacatcca ctgggtccga 181 caggcccctg gcaagggcct ggaatgggtg gcccggatct accccaccaa cggctacacc 241 agatacgccg actccgtgaa gggccggttc accatcfcccg ccgacacctc caagaacacc 301 gcctacctgc agatgaactc cctgcgggcc gaggacaccg ccgtgtacta ctgctccaga 361 tggggaggcg acggcttcta cgccatggac tactggggcc agggcaccct ggtcaccgtg

421 ctccgcta gc

Nucleotide sequence of Anti-HER2/neu light chain variable region

1 gcggccgcca tggaatccca gacccaggtg ctgatctccc tgctgttctg ggtgtccggc

61 acctgtggcg acatccagat gacccagtcc ccctccagcc tgtccgcctc tgtgggcgac

121 agagtgacca tcacctgtcg ggcctcccag gacgtgaaca ccgccgtggc ctggtatcag

181 cagaagcccg gcaaggcccc caagctgctg atctactccg cctccttcct gtactccggc

241 gtgccctccc ggttctccgg ctctagatcc ggcaccgact ttaccctgac catctccagc

301 ctgcagcccg aggacfctcgc cacctactac tgccagcagc actacaccac cccccccacc

361 tttggccagg gcaccaaggt ggaaatcaag cggaccgtgg ccgctccctc cgtgttcatc

421 cccaccct ccgacgagca gctg

Nucleotide sequence of Anti-EGFR1 heavy chain constant region with linker:

1 gctagcacca agggcccctc cgtgtttccc ctggccccct ccagcaagtc cacctctggc

61 ggcaccgccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc

121 tggaactctg gcgctctgac ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc

181 ggcctgtact ccccgtcctc cgtcgtgacc gtgccctcca gctctctggg cacccagacc

241 tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagcg ggtggaaccc

301 aagtcctgcg acaagaccca cacctgtccc ccctgccctg cccctgaact gctgggaggc

361 ccttccgtgt tcctgttccc cccaaagccc aaggacaccc tgatgatctc ccggaccccc

421 gaagtgacct gcgtggtggt ggacgtgtcc cacgaggacc ctgaagtgaa gttcaattgg

481 tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ccagagagga acagtacaac

541 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaaa

601 gagtacaagt gcaaggtgtc caacaaggcc ctgcctgccc ccatcgaaaa gaccatctcc

661 aaggccaagg gccagccccg cgagcctcag gtgtacaccc tgccteccag ccgggacgag

721 ctgaccaaga accaggtgtc cctgacctgt ctggtcaagg gcttctaccc ctccgatatc

781 gccgtggaat gggagtccaa cggccagccc gagaacaact acaagaccac cccccctgtg

841 ctggactccg acggctcatt cttcctgtac tccaagctga ccgtggacaa gtcccggtgg

901 cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc

961 cagaagtccc tgtctctgag ccccggcaaa ggcggcggag gatctggcgg tggcggatca

1021 gsfcggag gatcc

Figure 17
18/65

Nucleotide sequence of Anti-EGFR1 heavy chain variable region

2017201572 07 Mar 2017

1 gcggccgcca tgaacttcgg cctgcggctg atcttcctgg tgctgaccct gaagggcgtg 61 cagtgccagg tgcagctgaa gcagtccgga cctggcctgg tgcagccttc ccagtccctg

121 tccatcacct gtaccgtgtc cggcttctcc ctgaccaact acggcgtgca ctgggtccga 1B1 cagtccccag gcaagggcct ggaatggctg ggagtgattc ggagcggcgg caacaccgac 241 tacaacaccc ccttcacctc ccggctgtcc atcaacaagg acaactccaa gtcccaggtg 301 ttcttcaaga tgaactccct gcagtccaac gacaccgcca tctactactg cgccagagcc 361 ctgacctact atgactacga gttcgcctac tggggacagg gcaccctggt caccgtgtct 421 cgctagc

Nucleotide sequence of Anti-EGFR1 light chain variable region

1 gcggccgcca tggaatccca gacccaggtg ctgatctccc tgctgttctg ggtgtccggc 61 acctgtggcg acatcctgct gacccagtcc cccgtgatcc Cgtccgtgtc tcctggcgag

121 cgggtgtcct tctcctgccg ggcctcccag tccatcggca ccaacatcca ctggtatcag

181 cagcggacca acggctcccc tcggctgctg attaagtacg cctccgagtc tatctccggc

241 atcccctccc ggttctccgg ctctggctcc ggcaccgact tcaccctgtc catcaactcc

301 gtggaatccg aggatatcgc cgactactac tgccagcaga acaacaactg gcccaccacc

3S1 ttcggcgctg gcaccaagct ggaactgaag cggaccgtgg ccgctccctc cgtgttcatc

421 cccaccct ccgacgagca gctg

Nucleotide sequence of Anti-CTLA4 heavy chain variable region

1 gcggccgcca tgaacttcgg cctgcggctg atcttcctgg tgctgaccct gaagggcgtg

61 cagtgccagg tgcagctggt ggaatccggc ggaggcgtgg tgcagccLgg cagatccctg

121 agactgtcct gcgccgcctc cggcttcacc ttctccagct acaccatgca ctgggtccga

181 caggcccctg gcaagggcct ggaatgggtc accttcatca gctacgacgg caacaacaag

241 tactacgccg actccgtgaa gggccggttc accatctccc gggacaactc caagaacacc

301 ctgtacctgc agatgaactc cctgcgggcc gaggacaccg ccatctacta ctgcgcccgg

361 accggcfcggc tgggcccttt tgattactgg ggccagggca ccctggtcac cgtgtcctcc

421 tagc

Nucleotide sequence of Anti-CTLA4iight chain variable region

1 gcggccgcca tggaatccca gacccaggtg ctgatctccc tgctgttctg ggtgtccggc 61 acctgtggcg agatcgtgct gacccagtcc cccggcaccc tgtctctgag ccctggcgag

121 agagccaccc tgtcctgcag agcctcccag tccgtgggct cctcctacct ggcttggtat 181 cagcagaagc ccggccaggc ccctcggctg ctgatctacg gcgctttctc tcgggccacc 241 ggcatccctg accggttctc tggctccggc tccggcaccg acttcaccct gaccatctcc

301 cggctggaac ccgaggactt cgccgtgtac tactgccagc agtacggctc ctccccctgg

361 acctttggcc agggcaccaa ggtggaaatc aagcggaccg tggccgctcc ctccgtgttc

421 cttcccac cctccgacga gcagctg

Figure 18
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2017201572 07 Mar 2017

Nucleotide sequence of Anti CD2C tgG1 molecule:

1 gctagcacaa agggccctag tgtgtttcct ctggctccct cttccaaatc cacttctggt

61 ggcactgctg ctctgggatg cctggtgaag gattactttc ctgaacctgt gactgtctca

121 tggaactctg gtgctctgac ttctggtgtc cacactttcc ctgctgtgct gcagtctagt

181 ggactgtact ctctgtcatc tgfcggtcact gtgccctctt catctctggg aacccagacc

241 tac.atttgta atgtgaacca caaaccatcc aacactaaag tggacaaaaa agccgaaccc

301 aaatcctgtg acaaaaccca cacctgccca ccttgtcctg cccctgaact gctgggagga

361 ccttctgtgt ttctgttccc accaaaacca aaagataccc tgatgatctc tagaacccct

421 gaggtgacat gtgtggtggt ggatgtgtct catgaggacc ctgaggtcaa atttaattgg

481 tacgtcgatg gagtggaagt ccacaatgcc aaaaccaagc ctagagagga acagtacaat

541 tcaacctaca gagtcgtcag tgtgctgact gtgctgcatc aggattggct gaatggcaag

601 gaatacaagt gtaaagtctc aaacaaggcc ctgcctgctc caattgagaa aacaatctca

661 aaggccaagg gacagcctag ggaaccccag gtctacaccc tgccaccttc acgcgacgaa

721 ctgaccaaaa acoaggtgtc cctgacatgc ctggtcaaag gcttctaccc ttctgacart

781 gctgtggagt gggagtcaaa tggacagcct gagaacaact acaaaacaaa cccccctgtg

841 ctggattctg atggctcttt ctttctgtac tccaaactga ctgtggacaa gtctagatgg

901 cagcagggga atgtcttttc ttgctctgtc atgcatgagg ctctgcataa ccactacact

961 cagaaatccc tgtctctgtc tcccgggaaa ggcggcggag gatctggcgg aggcggttct

1021 ggtggtggcg gatcc

Nucleotide sequence of Anti-CD20 heavy chain variable region

1 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc

61 cagtgtcagg tgcagctgca gcagcctggt gccgagctcg tgaaacctgg cgcctccgtg

121 aagatgtcct gcaaggcctc cggctacacc ttcaccagct acaacatgca ctgggtcaag

181 cagacccccg gcagaggcct ggaatggatc ggcgctatct accccggcaa cggcgacacc

241 tcctacaacc agaagttcaa gggcaaggcc accctgaccg ccgacaagtc ctcttccacc

301 gcctacatgc agctgtcctc cctgacctcc gaggactccg ccgtgtacta ctgcgcccgg

361 tctacctact acggcggcga ctggtacttc aacgtgtggg gcgctggcac caccgtgacc

421 gtgtctgctg ctagc

Nucleotide sequence of Anti-CD20 light chain variable region

1 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc

61 cagtgtcaga tcgtgctgtc ccagtcccct gccatcctgt ctgctagccc tggcgagaaa

121 gtgacaatga cctgccgggc ctcctcctcc gtgtcctaca tecactggtt ccagcagaag

181 cccggctcca gccccaagcc ttggatctac gccacctcca acctggcctc tggcgtgcca

241 gtgcggtttt ccggctctgg ctctggcacc tcctaccccc tgaccatctc tcgggtggaa

301 gccgaggatg ccgccaccta ctactgccag cagtggacca gcaacccccc cacatttggc

361 ggaggcacca agctggaaat caagcggacc gtggcggcgc cctct

Figure 19
20/65

2017201572 07 Mar 2017

Nucleotide sequence of 4-1 BB.

1 ggatccgcct gtccttgggc cgtgtccggc gctagagcct ctcctggctc tgccgcctcc

61 cccagactga gagagggccc tgagctgtcc cctgacgatc ctgccggcct gctggacctg

121 agacagggca tgtttgccca gctggtggcc cagaacgtgc tgctgatcga cggccccctg

1S1 tcctggtact ctgatcctgg cctggccggc gtgtccctga ccggcggact gtcctacaaa

241 gaggacacca aagaactggt ggtggccaag gctggcgtgt actacgtgtt ctttcagctg

301 gaactgcggc gggtggtggc cggcgagggc tctggatctg tgcccctggc cctgcatctg

361 cagcccctga gatctgccgc tggcgccgct gctctggccc tgacagtgga tctgcctcct

421 gcctcctccg aggcccggaa ctccgcattc gggtttcagg gccggctgct gcacctgtct

431 gctggccaga gactgggagt gcatctgcac accgaggcca gagccagaca cgcctggcag

541 ctgacccagg gcgctaccgt gctgggcctg ttcagagtga cccccgagat cccagccggc

601 ctgcccagcc ctagatccga gtgataagct t

Nucleotide sequence of Anti-IL6R heavy chain:

1 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 61 cagtgtcagg tgcagctgca ggaatctggc cctggactcg tgcggccttc ccaaaccctg

121 tctctgacct gtaccgtgtc cggctactcc atcacctccg accacgcctg gtcttgggtg 181 cgacagcctc ctggcagagg cctggaatgg atcggctaca tctcctactc cggcatcacc 241 acctacaacc ccagcctgaa gtccagagtg accatgctgc gggacacctc caagaaccag 301 ttctccctgc ggctgtcctc cgtgaccgct gctgataccg ccgtgtacta ctgcgccaga 361 tctctggcca ggaccaccgc catggattac tggggccagg gctccctcgt gaccgtgtcc 421 tctgctagca ccaagggccc ctccgtgttc cctctggccc cttcctctaa atctacctct 461 ggcggcaccg cagctctggg ctgcctcgtg aaggactact tccccgagcc cgtgacagtg 541 tcttggaact ctggcgccct gacctccggc gtgcacacct ttccagctgt gctgcagtcc 601 tccggcctgt actccctgcc cagcgtcgtg actgtgccct cctcatctct gggcacccag 661 acctacatct gcaacgtgaa ccacaagccc tccaacacca aggtggacaa gaaggCggaa 721 cccaagtcct gcgacaagac ccacacctgt cccccttgtc ctgcccctga actgctgggc 731 ggaccctctg tgttcctgct cccaccaaaa ccgaaagaca ccctgatgat ctcccggacc 841 cccgaagtga actgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 901 tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 961 aactccacct accgggtggt gtccgtgctg accgtgctgc accaggattg gctgaacggc

1021 aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatcga aaagaccatc 1081 tccaaggcca agggccagcc acgggaaccc caggtgtaca cactgccccc tagccgcgac 1141 gagctgacca agaatcaggt gtccctgaca tgcctcgtga aaggcttcta cccctccgat 1201 atcgccgtgg aatgggagcc caacggccag cctgagaaca actacaagac caccccccct 1261 gtgctggact ccgacggccc attcttcctg tactcaaagc tgacagtgga caagtcccgg 1321 tggcagcagg gcaacgtgzt ctcctgctcc gtgatgcacg aggccctgca caaccactac 1381 acccagaagt ccctgtccct gagccccggg aaaggcggcg gaggatctgg cggaggcggt 1441 tctggtggtg gcggatcc

Figure 20
21/65

Nucleotide sequence of Anti-IL6R light chain variable region:

2017201572 07 Mar 2017

1 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc

61 cagtgtgaca tccagatgac ccagtccccc tccagcctgt ctgcctctgt gggcgacaga

121 gtgaccatca cctgtcgggc ctcccaggac atctcctcct acctgaactg gtatcagcag

181 aagcccggca aggcccccaa gctgctgatc tactacacct cccggctgca ctccggcgtg

241 ccctctagat tttccggctc tggctccggc accgacttta ccttcaccat cagctccctg

301 cagcccgagg atatcgccac ctactactgc cagcaaggca acaccctgcc ctacaccttt

361 ggccagggca ccaaggtgga aatcaagcgg accgtggcgg cgccc

Nucleotide sequence of Anti-4-1BB heavy chain

1 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc

61 cagtgtcagg tgcagctgca gcagtgggga gctggactgc tgaagccctc cgagacactg

121 tctctgacct gcgctgtgta cggcggctcc ttctccggct actactggtc ctggattcgg

181 cagtcccctg agaagggcct ggaatggatc ggcgagatca accacggcgg ctacgtgacc

241 tacaacccca gcctggaatc cagagtgacc atctccgtgg acacctccaa gaaccagttc

301 tccctgaagc tgtcctccgt gaccgccgct gataccgccg tgtactactg cgccagagac

361 tacggccctg gcaactacga ctggtacttc gacctgtggg gcagaggcac cctcgtgacc

421 gtgtcctctg ctagcaccaa gggcccctcc gtgtttcctc tggccccttg ctcacgctcc

481 acctccgaat ctaccgccgc tctgggctgc ctcgtgaagg actacttccc cgagcccgtg

541 actgtgtctt ggaactctgg cgccctgacc tccggcgtgc acacctttcc agctgtgctg

601 cagtcctccg gcctgtactc cctgtccagc gtcgtgacag tgccctccag ctctctgggc

661 accaagacct acacctgtaa cgtggaccac aagccctcca acaccaaggt ggacaagcgg

721 gtggaatcta aatacggccc tccctgccct ccttgcccag cccctgaatt tctgggcgga

781 ccttccgtgt tcctgttccc cccaaaaccc aaggacaccc tgatgatctc ccggaccccc

341 gaagtgacct gcgtggtggt ggatgtgtcc caggaagatc ccgaggtgca gttcaattgg

901 tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ctagagagga acagttcaac

961 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggattggct gaacggcaaa

1021 gagtacaagt gcaaggtgtc caacaagggc ctgcccagct ccatcgaaaa gaccatcagc

1081 aaggccaagg gccagccccg ggaaccccag gtgtacacac tgcctccaag ccaggaagag

1141 atgaccaaga atcaggtgtc cctgacctgt ctcgtgaaag gcttctaccc ctccgatatc

1201 gccgtggaat gggagtccaa cggccagcct gagaacaact acaagaccac cccccctgtg

1261 ctggactccg acggcagctt cttcctgtac tctcgcctga ccgtggacaa gtcccggtgg

1321 caggaaggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc

1381 cagaagtccc tgtccctgtc tctggggaaa ggcggcggag gatctggcgg aggcggttct

1441 ggtggtggcg gaLqc

Nucleotide sequence of Anti-4-1 BB Eight chain variable region

1 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc

61 cagtgtgaga tcgtgctgac ccagtctcct gccaccctgt ctctgagccc tggcgagaga

121 gctaccctgt cctgccgtcc ctcccaatcc gtgtcctctt acctggcctg gtatcagcaa

181 aagcccggcc aggctccccg gctgctgatc tacgatgcct ccaatagagc caccggcatc

241 cctgccagat tctccggctc tggctctggc accgacttta ccctgaccat ctcctctctg

301 gaacccgagg acttcgccgt gtactactgc cagcagcggt ccaactggcc tcccgccctg

361 acatttggcg gaggcaccaa ggtggaaatc aagcggaccg tggcggcgcc c

Figure 21
22/65

2017201572 07 Mar 2017 _£sdU£fi£L Non-Reduced

Molecular wt markers

Light chain _r

Figure 22
23/65

2017201572 07 Mar 2017

Figure 23
24/65

2017201572 07 Mar 2017

Concentration (pg/ml)

--B-- Anti-HER2-TGF£RI! Refrozen —S3— Ant-EGFRf -: GFSRIi

Anti-EGFR1-TGFpRi! Refrozen

Bmab 100(Avastin)

TGFpRII-Fc

Anti-HER2-TGFBRII’2-8^C

Inhibition of BT474 Proliferation

BmablOO

Bmab200 —Anti-HhR2-rGrSR!l

-O— Buffer

Figure 24
25/65

2017201572 07 Mar 2017

Inhibition of A431 Proliferation «Ο BmablOOfAvastin) «*» AnS-cGFRl-1GF-3 3

Figure 25 Buffer •Cetuximafa
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28/65

2017201572 07 Mar 2017

Anti-EGFRl-4-lBB ADCC ASSAY 29112011

-*» Buffer Cetuximab Anti-EGFRl-TGFpRII Anti EGFR14-lBB purified

Figure 28
29/65

2017201572 07 Mar 2017

Anti-CTLA4-TGFpRII ELISA 15112011 (1:120,000 diln ) —·— BmablCO (Avastin) ·*· Anti-EG FR l-TGFp Fill

Antl-CTLA4-TGFpRli/2-8°C

Anti-CTLA4-TGFpR!l Frozen

Anti~CTLA4-TGFpRII /CTLA4 ELISA 09112011

-O-Anti-CTLA4-TGFpRll Frozen BmablOO (Avastin)

AntiCTLA4-TGFf5RII/2-8°C

Figure 29
30/65

2017201572 07 Mar 2017 • Anti-EGFRl-TGFpRII —6— PDLl-Fc

-0- Anti CTLA4 TGFpRII/2-S*C «Ο Anti-CTLA4TGFpRII Frozen

Concentration (fig/mij

Anti-EGFRl-TGFpRII

4-1BBL

Anti-EGFRl-4-lBB SUP2-8^SC

Anti-EGFR.l-4-lBB Purified

Figure 30
31/65

2017201572 07 Mar 2017

A Anti-EGFRi-4-lBB SUPERNATENT EGFR ELISA

-*-B mat, 100 {AvastinJ

-«“AMi-EGFRl-T^RII —Anti-EGFRl-4-lBB 2-8’C — — Anti-EGFRl-4-lBB frozen

Concentration (og/ml)

Concentration (ng/ni·]

Figure 31
32/65

2017201572 07 Mar 2017

Anti-EGFRl-PDl EGFR ELISA 04112011 i < BmablOOjAvastin)

-«-.Anti-EGFRI-TGFPRII —Anti-EGFRl-PDl 2-8’c —AntiEGFRl-PDl frozen

Concentration (yg/ml)

Figure 32

33/65

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Figure 45
46/65

2017201572 07 Mar 2017

Cantuzumab -TGFpRII fusion protein at LC constant region

Amino acid sequence of Cantuzumab heavy chain:

QVQLVQSGAEVKKPGETVKiSCKASDYTFTYYGMNWVKQAPGQGLKWMG

WIDTTTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYN

WYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP

SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDiAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Amino acid sequence of Cantuzumab light chain fusion protein:

DIVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIY

RMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGT

KLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ

SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF

NRGEC

GGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQL

CKFCDVRFSTCDNQKSCMSNCSITS1CEKPQEVCVAVWRKN

DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF

FMCSCSSDECNDNIIFSEEYNTSNPD

Figure 46

2017201572 07 Mar 2017
47/65

Cixutumumab -TGFpRIt fusion protein at LC constant region

Amino acid sequence of Cixutumumab heavy chain:

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGi

IPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLE

WSTQDHYYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG

CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ

TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD

TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY

RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP

SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF

LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Amino acid sequence of Cixutumumab light chain fusion protein:

SSELTQDPAVSVALGQTVRITCQGDSLRSYYATWYQQKPGQAPILVIYGENK

RPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCKSRDGSGQHLVFGGGTKL

TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK

AGVETTTPSKQSN N KYAASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTVAP

AECSGGGG5GGGG5GGGG5TIPPHVQKSVNNDMIVTDNNGAVKF

PQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVW

RKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPG

ETFFMCSCSSDECNDNIIFSEEYNTSNPD

Figure 47
48/65

2017201572 07 Mar 2017

Clivatuzumab -TGFpRII fusion protein at LC constant region

Amino acid sequence of Clivatuzumab heavy chain:

QVQLQQSGAEVKKFGASVKVSCEASGYTFPSYVLHWVKQAPGQGLEWIGYI

NPYN DGTQTN KKFKG KATLTRDTSINTAYM E LSRLRSDDTAVYYCARG FGGS

YGFAYNGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

TVSWNSGALTSGVNTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS

NTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNISRTPEVTC

WVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPiEKTiSKAKGQPREPQVYTLPPSREEMTKNQV

SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

WQQGNVFSCSVN H EALHN H YTQKSLSLSPG K

Amino acid sequence of Clivatuzumab light chain fusion protein:

DIQLTQSPSSLSASVGDRVTMTCSASSSVSSSYLYWYQQKPGKAPKLWIYSTS NLASGVPARFSGSGSGTDFTLTISSLQPEDSASYFCHQWNRYPYTFGGGTRLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYEAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSPR KADYEKH KVYACE VTHQG LSSPVTKSFN R

GECGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFP

QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWR

KNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE

TFFMCSCSSDECNDNIIFSEEYNTSNPD

Figure 48
49/65

Pritumumab-TGFpRII fusion protein at LC constant region

2017201572 07 Mar 2017

Amino acid sequence of Pritumumab heavy chain:

EVQLLESGGDLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSAI

TPSGGSTNYADSVKGRFTISRDNSQNTLYLQMNSLRVEDTAVYICGRVPYRST

WYPLYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

VTVSWNSG ALTSG VHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVN H KP

SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQG N VFSCS VM H EALH N H YTQKSLSLS PG K

Amino acid sequence of Pritumumab light chain fusion protein:

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWFQQKPGKAPKSLIYAASS

LHSKVPTQFSGSGSGTDFTLTISSLQPEDFATYYCLQYSIYPITFGGGTKVEIKR

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ

ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

GGGG5GGGG5GGGG5TIPPHVQKSVNNDMIVTDNNGAVKFPQL

CKFCDVRFSTCDNQKSCMSNCS1TSICEKPQEVCVAVWRKN

DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF

FMCSCSSDECNDNIIFSEEYNTSNPD

Figure 49
50/65

2017201572 07 Mar 2017

Cantuzumab HC-4-1BB and LC-TGFpRII fusion protein

Amino acid sequence of heavy chain-4-ΙΒΒ fusion protein:

QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMG

WIDTTTGEPTYAQKFQGR1AFSLETSASTAYLQ1KSLKSEDTATYFCARRGPYN

WYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP

SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKITPPVLDSDGSFFLYSKLTVDKSRW

QQG N VFSCSVM H EALH N HYTQKSLSLSPGKGGGGSGGGG5GGGGSACP

WAVSGAKASPGSAASPRLHFGPELSPOOPAGLLOLKQGM

FAQLVAQWLLWGPLSWSVPGLAGVSLrGGLSYKBVTK.

FLWAKAGVYYVFFQLFLH'R-y^AGFGSGSVSLALHLQPLHS

AA GAAA LALTVOLVPASStAKKSA PGPQGPLLH LSA GQK

LGVHLHTFAHAIZHAWQLrQGATVLGLFHVTPFIPAGLPS

PKSt

Amino acid sequence of Cantuzumab light chain fusion protein:

DIVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLUY

RMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGT

KLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ

SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF

NRGEC

GGGGSGGGGSGGGGSTlPPHVQKSVNNDMiVTDNNGAVKFPQL

CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN

DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF

FMCSCSSDECNDNIIFSEEYNTSNPD

Figure 50
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Cixutumumab HC-4-1BB and LC-TGFpRII fusion protein

2017201572 07 Mar 2017

Amino acid sequence of heavy chain-4-ΙΒΒ fusion protein:

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGI

IPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLE

WSTQDHYYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG

CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ

TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD

TLMiSRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY

RVVSVLTVLHQ.DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP

SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF

LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGG

GSGGGGSACHWAVS^AnASFQSAASHKLKEGPFLSHVVFA^

LLVL'HQ^MFAQLVAQ^y^WQHLSWS'DHQLAQVSLTQ

G LSYKFOTKFLVVA KA GVYYVffQ LELKWVA GFGSGSVS L

ALHLQPLRSAAGAAALALTVVLPPASSEAWSAFGFQGIlL

LH LSAGQR LQVH LH ΤΈΑ1ΖΑΗΗΑ WQLTQGA TVLGLfKVT

PEIPAGUPSPKSt

Amino acid sequence of Cixutumumab light chain fusion protein:

SSELTQDPAVSVALGQTVR1TCQGDSLRSYYATWYQQKPGQAPILVIYGENK

RPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCKSRDGSGQHLVFGGGTKL

TVLGQPKAAPSVTLFPPSSEELQANKATLVCLiSDFYPGAVTVAWKADSSPVK

AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP

AECSGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKF

PQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVW

RKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPG

ETFFMCSCSSDECNDNIIFSEEYNTSNPD

Figure 51
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Clivatuzumab HC-4-1BB and LC-TGFPRII fusion protein

2017201572 07 Mar 2017

Amino acid sequence of heavy chain -4-1BB fusion protein

QVQLQQSGAEVKKFGASVKVSCEASGYTFPSYVLHWVKQAPGQGLEWIGYi

N PYN DGTQTN KKFKG KATLTRDTSI NTAYM ELSRLRSDDTAVYYCARGFGGS

YGFAYNGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

TVSWNSGALTSGVNTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS

NTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNISRTPEVTC

VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV

5LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

WQQG NVFSCSVN H EALH N H YTQKSLS LSPG KGGGGSGGGGSGGGGSA C

PWAVSGARASPGSAASI^L^Gf^LSPVVPAGLL'DL'RQG

WAQLVAQNVLLWGPLSWSOPGLAGVSLTGGLSYKZVT

KtLWAKAGWVffQLtLHKWAGtGSGSVSLALHLQPLK

SAAGAAA LA LTVD LPPASSZAKttSA TGTQGK LLH LSA GQK

LGW LH TLAKAKHA WQLTQGATVLGLFKVrpLDPAGLPS

PKSt

Amino acid sequence of Clivatuzumab light chain fusion protein:

DIQLTQSPSSLSASVGDRVTMTCSASSSVSSSYLYWYQQKPGKAPKLWIYSTS NLASGVPARFSGSGSGTDFTLTISSLQPEDSASYFCHQWNRYPYTFGGGTRLE IKRTVAAPSVFI FPPSD EQLKSGTASVVCLLN N FYEAKVQWKVDN ALQSGNS QESVTEQDSKDSTYSLSSTLTLSPRKADYEKHKVYACEVTHQGLSSPVTKSFNR

GECGGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFP

QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWR

KNDENITLETVCHDPKLPYHDF1LEDAASPKCIMKEKKKPGE

TFFMCSCSSDECNDNIIFSEEYNTSNPD

Figure 52

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Pritumumab HC-4-1BB and LC-TGFBRil fusion protein

Amino acid sequence of heavy chain-4-ΙΒΒ fusion protein:

EVQLLESGG DLVQPGGSLRLSCAASG FTFSN YAMSWVRQAPG KG LEWVSAI

TPSGGSTNYADSVKGRFTiSRDNSQNTLYLQMNSLRVEDTAVYICGRVPYRST

WYPLYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP

SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQG N VFSCSVM Η E ALH N H YTQKSLSLS PG KGGGGSGGGGSGGGGSACP

WAVSGAUASPGSAASPULUFGPLLSPVVPAGLLVL'RQGM

FAQLVAQNVLLIVQrLSWYSVr0LA<SVSLr^GLSYK,WTK

ΈΙΛΑ/ΑKA GVYWFFQ LLLKRWA&EGSGSVSLA LULQPLKS

AAGAAA LA LTVOLPPASStAKMSA FGFQQKLLU LSA GQU

LGVULUrLAUAUUAWQLrQGATVLGLFUVrpEirA^LPS

PKSL

Amino acid sequence of Pritumumab light chain fusion protein:

DIQMTQSPSSLSASVGDRVTiTCRASQDISNYLAWFQQKPGKAPKSLIYAASS

LHSKVPTQFSGSGSGTDFTLTISSLQPEDFATYYCLQYSTYPITFGGGTKVEiKR

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ

ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

GGGGSGGGGSGGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQL

CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN

DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF

FMCSCSSDECNDNIIFSEEYNTSNPD

Figure 53
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Cantuzumab - HC-PD1 and LC-TGFf$RII fusion protein

Amino acid sequence of heavy chain-PDl fusion protein:

QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMG

WIDTrTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYN

WYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

VTVSWNSG ALTSG VHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVN H KP

SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPiEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSFGWf lospvkpwnpptfsfa ll wtegoma THcsFSKrsESfVLNWYm SFS^QrVKLAAFFEOHSQPGQOCTiFKVr^LP^K'DFHMSYYKA mmS^TYLCGAISLAFKAQIKESLHAELKVrEKHAEVFTAHFSPS -pKPAQQFQTLV

Amino acid sequence of Cantuzumab light chain fusion protein:

DfVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIY

RMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGT

KLELKRTVAAPSVF1FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ

SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF

NRGEC ggggsggggsggggstipphvqksvnndmivtdnngavkfpql

CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN

DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF

FMCSCSSDECNDNIIFSEEYNTSNPD

Figure 54

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Cixutumumab HC-PD1 and LC-TGFpRII fusion protein

Amino acid sequence of heavy chain-PDl fusion protein:

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGI

IPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLE

WSTQDHYYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG

CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ

TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD

TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY

RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP

SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF

LYSKLTVDKSR WQQG N VFSCSVM H EALHN H YTQKSLSLSPG KGGGGSGGG

GSGGGGSPGWrLVSPO'iZPWNPprrSPA LLWTEGOk'A TfrCSfiS^T mjmMSVVHAmhrDSGTYLCGAISLAPKAQIKESLPAELKVr^KP

AZVPTAHPSPSPRPAGQFQTLV

Amino acid sequence of Cixutumumab light chain fusion protein:

SSELTQDPAVSVALGQTVRITCQGDSLRSYYATWYQQKPGQAPILVJYGENK

RPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCKSRDGSGQHLVFGGGTKL

TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK

AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP

AECSGGGG5GGGG5GGGGSTIPPHVQKSVNNDMIVTDNNGAVKF

PQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVW

RKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPG

ETFFMCSCSSDECNDNIIFSEEYNTSNPD

Figure 55

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Clivatuzumab HC-PD1 and LC-TGFpRII-fusion protein

Amino acid Amino acid sequence of heavy chain-PDl fusion protein:

QVQLQQSGAEVKKFGASVKVSCEASGYTFPSYVLHWVKQAPGQGLEWIGYi

NPYNDGTQTNKKFKGKATLTRDTSINTAYMELSRLRSDDTAVYYCARGFGGS

YGFAYNGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

TVSWNSGALTSGVNTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVN H KPS

NTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNISRTPEVTC

VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAP1EKTISKAKGQPREPQVYTLPPSREEMTKNQV

SLTCLVKGFYPSD1AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

WQQG NVFSCSVN HEALH N HYTQKSLS LSPG KGGGGSGGGGSGGGGSPG

WJWSPOPPWNPPTFSPA LLWTEGONA rpTCS'FSNTSPSFVLNWY

IZAmNVSGrYLCGAISLAPKAQIKFSL'R.AFL'R.VrEmAPVprAHPS

PSPRPAGQFQTLV

Amino acid sequence of Clivatuzumab light chain fusion protein:

DIQLTQSPSSLSASVGDRVTMTCSASSSVSSSYLYWYQQKPGKAPKLWIYSTS

NLASGVPARFSGSGSGTDFTLTISSLQPEDSASYFCHQWNRYPYTFGGGTRLE

IKRTVAAPSVF1FPPSDEQLKSGTASVVCLLNNFYEAKVQWKVDNALQSGNS

QESVTEQDSKDSTYSLSSTLTLSPRKADYEKHKVYACEVTHQGLSSPVTKSFNR

GECGGGGSGGGGSGGGGSTIPPHVQKSVNNDMiVTDNNGAVKFP

QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWR

KNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGE

TFFMCSCSSDECNDNIIFSEEYNTSNPD

Figure 56

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Pritumumab HC-PD1 and LC-TGFpRif fusion protein

Amino acid Amino acid sequence of heavy chain-PDl fusion protein:

EVQLLESGGDLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSAI

TPSGGSTNYADSVKGRFTISRDNSQNTLYLQMNSLRVEDTAVYICGRVPYRST

WYPLYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP

SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPIEKTiSKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGGGGGSGGGGSiWF

SPShl^rVKLAAmmSQr^QOC'R.r'R.VTQLi’^KVFHMSVVKA

KHNVSGr/LCQAISLAPKAQIKESURAELKVTEIlRAEVPTAHPSPS

PRPAGQFQTLV

Amino acid sequence of Pritumumab light chain fusion protein:.

DIQMTQSPSSLSASVGDRVTiTCRASQDISNYLAWFQQKPGKAPKSLIYAASS

LHSKVPTQFSGSGSGTDFTLTISSLQPEDFATYYCLQYSTYPITFGGGTKVEiKR

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ

ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ggggsggggsggggstipphvqksvnndmivtdnngavkfpql

CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKN

DENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETF

FMCSCSSDECNDNIIFSEEYNTSNPD

Figure 57

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Cantuzumab HC-TGFpRII-4-lBB fusion protein

Amino acid sequence of heavy chain-TGFpRII-4’lBB fusion protein:

QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMG

W1DTTTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYN

WYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP

SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

Q.QGN VFSCSVM HEALHN H YTQKS LSLSPG KGGGGSGGGGSGGGGST1P P

HVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKS

CMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY

HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFS

EEYNTSNPDepkscdk4CW4VS<^ft45P<7S44SPRLR£GP flspoopagllolrqgmfaqlvaqwllivgplswysvp ^LAQYSLr^LSYK^VrKFLWAKA^VYYVrFQLFLnKVVA GEGSGSVSLA LHLQPLKSAAGAAA LA LTVOLPPASSFAW SAF^r^HLLHLSAGQKL^VHLHreAKAKHAWQLTQ^A TVLGLFKVTttlPAGUPSPKSt

Amino acid sequence of light chain

DiVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIY

RMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGT

KLELKRTVAAPSVFiFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ

SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF

NRGEC

Figure 58

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Cixutumumab HC-TGFpRil-4-lBB fusion protein

Amino acid sequence of heavy chain-TGF3RII-4-lBB fusion protein:

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGI

IPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLE

WSTQDHYYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG

CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ

TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD

TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY

RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP

SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF

LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGG5GGG

G5GGGGSTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVR

FSTCDNQKSCMSNCSITSiCEKPQEVCVAVWRKNDENITLET

VCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSS

DECNDNIIFSEEYNTSNPDepksctf/cACPWVS^A7M5mS/l

ASmLKt^LSPVVPA^LLVLKQQMrAQLVAQWLU'D ^LSWSVPQLAGVSLr^GLSYK^OTXFLWAKAGWYVFF

QLtLRKVVA GtGSGSVSLA LH LOfpLKSAA GAAA LA LTVVL

PPASS^AH^SAFGFQGKLLHLSAGQKLGVHLHrEAKA'R.H

AWQLTQGATVLGLFWTVLIPAGLPSPKS^

Amino acid sequence of light chain:

SSELTQDPAVSVALGQWRITCQGDSLRSYYATWYQQKPGQAPILVIYGENK

RPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCKSRDGSGQHLVFGGGTKL

TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK

AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP

AECS

Figure 59
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Clivatuzumab HC-TGFPRII-4-lBB fusion protein

Amino acid sequence of heavy chain-TGFPRIi-4-lBB fusion protein:

QVQLQQSGAEVKKFGASVKVSCEASGYTFPSYVLHWVKQAPGQGLEWIGYI

N PYNDGTQTN KKFKG KATLTRDTSI NTAYM E LSRLRSDDTAVYYCARG FGGS

YGFAYNGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

TVSWNSGALTSGVNTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS

NTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNISRTPEVTC

VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV

SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

WQQGNVFSCSVNHEALHNHYTQKSLSLSPGKGGGGSGGGG5GGGGSTIP

PHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQK

SCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLP

YHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF

SEEYmSHPDepkscdkACPWAVS^AHASPGSAASPKLPEG

PELSPVOPAGLLVLRQGMfAQLVAQNVLLWGPLSWYSV rQLAGVSLT(^QLSYKEVTXELWAT<AGVYYVrFQLELKHW

AGEGSGSVSLALHLQPLKSAAWAALALTVDLPPASStAR toSAFGFQGKLLHLSAGC&LGVHLHTEARARHAWQLTQG

ATVLGLfWTPEIPAGLPSPHSE

Amino acid sequence of light chain:

DIQLTQSPSSLSASVGDRVTMTCSASSSVSSSYLYWYQQKPGKAPKLWIYSTS

NLASGVPARFSGSGSGTDFTLTISSLQPEDSASYFCHQWNRYPYTFGGGTRLE

IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYEAKVQWKVDNALQSGNS

QESVTEQDSKDSTYSLSSTLTLSPRKADYEKHKVYACEVTHQGLSSPVTKSFNR

GEC

Figure 60
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Pritumumab HC-TGFpRII-4-lBB fusion protein

Amino acid sequence of heavy chain-TGFpRt(-4-lBB fusion protein:

EVQLLESGGDLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSAI

TPSGGSTNYADSVKGRFTiSRDNSQNTLYLQMNSLRVEDTAVYICGRVPYRST

WYPLYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP

SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPfEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQG N VFSCSVMH EALH N H YTQKSLSLSPG KGGGG5GGGG5GGGG5T1 PP

HVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKS

CMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY

HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNI1FS

EEYHJSHPDepkscdkACrWAVSGAKASPGSAASPHLH^H

ELSPOOPAGLLVLRQGMFAQLVAQNVLLIOGPLSWYSVP

GLAGVSLTGGLSYKZVTKZLWAKAGVYYVFFQLtLRRWA

GtGSGSVSLA LH LQPLKSAA GAAA LA LTVOLPPASStAKM

SAFGFQGKLLHLSAGQKLGVHLUTLAHAKHAWQLTQGA rVLGLHHVrpElPAGLHSrKSE

Amino acid sequence of light chain:

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWFQQKPGKAPKSLIYAASS

LHSKVPTQFSGSGSGTDFTLTISSLQPEDFATYYCLQYSTYPITFGGGTKVEIKR

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ

ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Figure 61

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Cantuzumab HC-TGFpRII-PDl fusion protein

Amino acid sequence of heavy chain-TGFPRILPDl fusion protein:

QVQLVQSGAEVKKPGETVKISCKASDYTFTYYGMNWVKQAPGQGLKWMG

WIDTTTGEPTYAQKFQGRIAFSLETSASTAYLQIKSLKSEDTATYFCARRGPYN

WYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

VTVSWNSGALTSG VHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVN H KP

SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPiEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGG5GGGG5GGGGSTIPP

HVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKS

CMSNCSITS1CEKPQEVCVAVWRKNDENITLETVCHDPKLPY

HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFS

EEYNTSN POepkscdkPGWfLVSPm PWWPPTfSPA LLWTEGV

KAELKVTTKHAEVPTAHPSPSPKPA^QfQTLV

Amino acid sequence of light chain:

DIVMTQSPLSVPVTPGEPVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIY RMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCLQHLEYPFTFGPGT KLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRG EC

Figure 62
63/65

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Cixutumumab HC-TGFpRII-PDl fusion protein

Amino acid sequence of heavy chatn-TGFPRIi-PDl fusion protein:

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGI

IPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAPLRFLE

WSTQDHYYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG

CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ

TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD

TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY

RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP

SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF

LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGG5GGG

GSGGGGSTIPPHVQKSVNNDMiVTDNNGAVKFPQLCKFCDVR

FSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLET

VCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSS

DECNDNIIFSEE YNTS N PDepkscdkp^WfLVSFDRP WWPPTFS

PALLWTE^V^ArFrcSFS^rSFSJVi^YTiMSPSF}QrVKLAAfPF

OHSQ^GQVCKFKVTQLPbJG'R.VFHMSWRAKW'DSGTYLCGAlSL

APKAQIKESLlZAFLKVVEnKAFVrrAHPSFSFIZPA^QrLV

Amino acid sequence of light chain:

SSELTQDPAVSVALGQTVRITCQGDSLRSYYATWYQQKPGQAPILVIYGENK RPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCKSRDGSGQHLVFGGGTKL TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK AG VETTTPSKQS N N KYAASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTVAP AECS

Figure 63
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Clivatuzumab HC-TGFpRII-PDl fusion protein

Amino acid sequence of heavy chafn-TGF£RII-PDl fusion protein:

QVQLQQSGAEVKKFGASVKVSCEASGYTFPSYVLHWVKQAPGQGLEWIGY!

Ν PYN DGTQTN KKFKGKATLTRDTSI NTAYM E LSRLRSDDTAVYYCARGFGGS

YGFAYNGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

TVSWNSGALTSGVNTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS

NTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLNISRTPEVTC

VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV

SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

WQQGNVFSCSVNHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGG5TIP

PHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQK

SCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLP

YHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIF

SEE YNTS N PDepkscdkP(jWFLOSFVKP\VWFprFSf’ALLY\/rE(jO bMrrrCSFSNTSESfVl^WYfZMSPSNQTVKLAAITEOnSC^PQQOC

PAELlZVTEmAEVprAHPSPSPriPAG^FQrLV

Amino acid sequence of light chain:

DIQLTQSPSSLSASVGDRVTMTCSASSSVSSSYLYWYQQKPGKAPKLWIYSTS

NLASGVPARFSGSGSGTDFTLTISSLQPEDSASYFCHQWNRYPYTFGGGTRLE

IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYEAKVQWKVDNALQSGNS

QESVTEQDSKDSTYSLSSTLTLSPRKADYEKHKVYACEVTHQGLSSPVTKSFNR

GEC

Figure 64

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Pritumumab HC-TGFpRII-PDl fusion protein

Amino acid sequence of heavy chain-TGFpRIi-PDl fusion protein:

EVQLLESGGDLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSAi

TPSGGSTNYADSVKGRFTISRDNSQNTLYLQMNSLRVEDTAVYICGRVPYRST

WYPLYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP

VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP

SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSTIPP

HVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKS

CMSNCSiTSICEKPQEVCVAVWRKNDENITLETVCHDPKLPY

HDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFS

EEYNTSN PDepkscdkPGWfLVSPm PWNPPTfSPA LLVVTEGV

NATFTCSFStirSESFVLNWYmS’PSNQrOKLAAFPEOKSQPGQ'DC

IZFNVrQLPNCIZOFPMSWHAKK^DSCrYLCCAISLA'PKAOrKESL

M^LKvrenKAWprAHrsrsrKrAGQrorLv

Amino acid sequence of light chain:

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWFQQKPGKAPKSLiYAASS

LHSKVPTQFSGSGSGTDFTLTISSLQPEDFATYYCLQYSTYPITFGGGTKVEIKR

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ

ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFIMRGEC

Figure 65

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3105559_l.txt

SEQUENCE LISTING <110> Biocon Limited

Govindappa, Nagaraj Sastry, Kedarnath Soares, Maria Melina <120> targeted/immunomodulatory fusion proteins and methods for making SAME <130> 014811.946.146DIV2 <150> 14/458,674 <151> 2014-08-13 <150> 13/799,409 <151> 2013-03-13 <150> 1690/CHE/2012 <151> 2012-04-30 <150> 1689/CHE.2012 <151> 2012-04-30 <160> 39 <170> Patentin version 3.5 <210> 1 <211> 449 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic construct

<400> 1 Glu 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Gly Ser Leu Arg Leu Ser 20 cys Ala Ala Ser Gly 25 Phe Asn lie Lys 30 Asp Thr Tyr ile His Trp Val 35 Arg Gln Ala Pro Gly 40 Lys Gly Leu 45 Glu Trp Val Ala Arg 50 ile Tyr Pro Thr Asn Gly Tyr Thr 55 Arg Tyr 60 Al a Asp Ser Val Lys 65 Gly Arg Phe Thr Ile 70 Ser Ala Asp Thr Ser 75 Lys Asn Thr Al a Tyr 80 Leu Gln Met Asn Ser 85 Leu Arg Ala Glu Asp 90 Thr Ala val Tyr Tyr 95 Cys Ser Arg Trp Gly Gly 100 Asp Gly Phe Tyr Ala 105 Met Asp Tyr T rp 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

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3105559_l.txt

Phe Pro 130 Leu Ala Pro Ser Ser Lys 135 Ser Thr Ser Gly Gly Thr Ala 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Al a Leu Thr Ser Gly val His Thr Phe Pro Ala Val 165 170 175 Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser val val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys val Asp Lys Lys val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr Hi s Thr cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys ASP Thr Leu Met lie 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val ASP Val Ser Hi s Glu 260 265 270 Asp Pro Glu val Lys Phe Asn Trp Tyr Val Asp Gly val Glu val Hi s 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg 290 295 300 val Val Ser Val Leu Thr Val Leu Hi s Gin Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu 325 330 335 Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gl n val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Gl u Glu Met Thr Lys Asn Gin Val Ser Leu 355 360 365 Thr cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Al a Val Glu Trp 370 375 380 Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro val

385 390 395 400

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3105559_l.txt

Leu Asp Ser Asp Gly Ser 405 Phe Phe Leu Tyr 410 Ser Lys Leu Thr Val 415 Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser cys Ser Val Met Hi s 420 425 430 Gl u Al a Leu His Asn Hi s Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly <210> 2 <211> 214 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic construct <400> 2

Asp Ile Gin Met Thr Gin Ser Pro Ser Ser 10 Leu Ser Ala Ser val 15 Gly 1 5 Asp Arg val Thr Ile Thr Cys Arg Al a Ser Gin Asp val Asn Thr Ala 20 25 30 Val Al a T rp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Al a Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Al a Thr Tyr Tyr cys Gin Gin Hi s Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gin Gly Thr Lys val Glu Ile Lys Arg Thr val Ala Al a 100 105 110 Pro Ser val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Al a 130 135 140 Lys Val Gin Trp Lys val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160 Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

Page 3

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3105559_l.txt

Ser Thr Leu Thr 180 Leu Ser Lys Al a Asp 185 Tyr Glu Lys Hi s Lys 190 Val Tyr Al a Cys Glu Val Thr Hi s Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys 210 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic construct <400> 3

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 15 10 15 <210> 4 <211> 137 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic construct

<400> 4 Thr lie Pro Pro His val Gln Lys Ser Val Asn Asn Asp Met Ile Val 1 5 10 15 Thr Asp Asn Asn Gly Al a Val Lys Phe Pro Gln Leu cys Lys Phe cys 20 25 30 Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn 35 40 45 Cys Ser ile Thr Ser lie cys Glu Lys Pro Gln Glu val Cys val Ala 50 55 60 val Trp Arg Lys Asn Asp Glu Asn lie Thr Leu Glu Thr Val Cys His 65 70 75 80 Asp pro Lys Leu Pro Tyr Hi s Asp Phe ile Leu Glu Asp Al a Al a Ser 85 90 95 Pro Lys cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe 100 105 110 Met cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser 115 120 125

Page 4

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31O5559_l.txt

Glu Glu Tyr 130 Asn Thr Ser Asn 135 Pro Asp <210> 5 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 5 Gln 1 Val Gln Leu Lys 5 Gln Ser Gly Pro Gly 10 Leu val Gln Pro Ser 15 Gln Ser Leu Ser lie 20 Thr Cys Thr Val Ser 25 Gly Phe Ser Leu Thr 30 Asn Tyr Gly Val Hi s 35 Trp Val Arg Gln Ser 40 Pro Gly Lys Gly Leu 45 Glu Trp Leu Gly Val Ile 50 Trp Ser Gly Gly 55 Asn Thr Asp Tyr Asn 60 Thr Pro Phe Thr Ser 65 Arg Leu Ser lie Asn 70 Lys Asp Asn Ser Lys 75 Ser Gln val Phe Phe 80 Lys Met Asn Ser Leu 85 Gln Ser Asn Asp Thr 90 Al a Ile Tyr Tyr cys 95 Al a Arg Ala Leu Thr 100 Tyr Tyr Asp Tyr Glu 105 Phe Al a Tyr Trp Gly 110 Gln Gly Thr Leu Val 115 Thr Val Ser Al a Ala 120 Ser Thr Lys Gly Pro 125 Ser Val Phe Pro Leu Ala 130 Pro Ser Ser Lys 135 Ser Thr Ser Gly Gly 140 Thr Ala Ala Leu Gly 145 Cys Leu val Lys Asp 150 Tyr Phe Pro Glu Pro 155 val Thr Val Ser T rp 160 Asn Ser Gly Ala Leu 165 Thr Ser Gly Val His 170 Thr Phe Pro Al a Val 175 Leu Gln Ser Ser Gly 180 Leu Tyr Ser Leu Ser 185 Ser Val Val Thr val 190 Pro Ser Ser Ser Leu 195 Gly Thr Gln Thr Tyr 200 ile cys Asn Val Asn 205 Hi s Lys Pro Ser Asn Thr 210 Lys Val Asp Lys 215 Arg val Glu Pro Lys 220 Ser Cys Asp Lys

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31O5559_l.txt

Thr 225 Hi s Thr Cys Pro Pro 230 Cys Pro Al a Pro Glu 235 Leu Leu Gly Gly Pro 240 Ser val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met ile Ser 245 250 255 Arg Thr Pro Glu Val Thr cys val val Val Asp Val Ser Hi s Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr val Asp Gly Val Glu val Hi s Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg val 290 295 300 val Ser Val Leu Thr Val Leu Hi s Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Al a Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Al a Lys Gly Gin Pro Arg Glu Pro Gin val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365 cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Al a Val Glu T rp Glu 370 375 380 Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 ASP Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gin Gin Gly Asn val Phe Ser Cys Ser val Met Hi s Glu 420 425 430 Al a Leu Hi s Asn Hi s Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445 <210> 6 <211> 214 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic construct <400> 6

Asp Ile Leu Leu Thr Gin Ser Pro val Ile Leu Ser Val Ser Pro Gly 15 10 15

Page 6

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2017201572 10 Mar 2017

Glu Arg val Ser 20 Phe Ser cys Arg Ala Ser 25 Gln Ser Ile Gly 30 Thr Asn lie Hi s T rp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser lie Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser val Glu Ser 65 70 75 80 Glu Asp lie Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Al a 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Al a Asp Tyr Glu Lys Hi s Lys Val Tyr 180 185 190 Al a cys Glu Val Thr Hi s Gln Gly Leu Ser Ser Pro val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu cys

210 <210> 7 <211> 447 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic construct <400> 7 Gln Val 1 Gln Leu val Glu Ser Gly Gly Gly val val 5 10 Gln Pro Gly Arg 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Page 7

3105559_l.txt

2017201572 10 Mar 2017

20 25 30

Thr Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Phe lie Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Al a Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Al a Glu Asp Thr Al a lie Tyr Tyr Cys 85 90 95 Al a Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Leu val Thr Val Ser Ser Al a Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Al a Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Al a Leu Gly 130 135 140 cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr val Ser T rp Asn 145 150 155 160 Ser Gly Al a Leu Thr Ser Gly val Hi s Thr Phe Pro Al a val Leu Gin 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser cys Asp Lys Thr 210 215 220 Hi s Thr cys Pro Pro cys Pro Al a Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys val Val Val Asp val Ser Hi s Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

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31O5559_l.txt

Ser val 305 Leu Thr Val Leu 310 Hi s Gln Asp Trp Leu Asn 315 Gly Lys Glu Tyr 320 Lys cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser val Met Hi s Glu Ala 420 425 430 Leu Hi s Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445 <210> 8 <211> 215 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic construct <400> 8 Glu lie Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Al a Thr Leu Ser Cys Arg Al a Ser Gln Ser val Gly Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 ile Tyr Gly Al a Phe Ser Arg Al a Thr Gly ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Al a Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

Page 9

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2017201572 10 Mar 2017

85 90 95 Trp Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg Thr Val Ala 100 105 110 Al a Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser 115 120 125 Gly Thr Al a Ser val val cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Al a Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser 145 150 155 160 Gl n Glu Ser val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys Hi s Lys val 180 185 190 Tyr Al a Cys Glu val Thr His Gin Gly Leu Ser Ser Pro val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys

210 215 <210> 9 <211> 205 <212> PRT <213> Artificial Sequence

<220> <223> <400> Synthetic construct Al a Arg Ala Ser Pro Gly Ser Ala 9 Pro Trp Ala 5 Val Ser Gly Al a 1 cys 10 15 Al a Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro 20 25 30 Al a Gly Leu Leu Asp Leu Arg Gin Gly Met Phe Al a Gin Leu val Al a 35 40 45 Gin Asn val Leu Leu Ile Asp Gly Pro Leu Ser T rp Tyr Ser Asp Pro 50 55 60 Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp 65 70 75 80 Thr Lys Glu Leu Val Val Al a Lys Ala Gly Val Tyr Tyr Val Phe Phe 85 90 95

Page 10

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2017201572 10 Mar 2017

Gln Leu Glu Leu 100 Arg Arg Val Val Ala Gly Glu Gly Ser 105 Gly 110 Ser val Ser Leu Ala Leu Hi s Leu Gln Pro Leu Arg Ser Al a Ala Gly Ala Al a 115 120 125 Ala Leu Al a Leu Thr val Asp Leu Pro Pro Ala Ser Ser Glu Al a Arg 130 135 140 Asn Ser Al a Phe Gly Phe Gln Gly Arg Leu Leu Hi s Leu Ser Ala Gly 145 150 155 160 Gln Arg Leu Gly val Hi s Leu Hi s Thr Glu Ala Arg Al a Arg His Al a 165 170 175 Trp Gln Leu Thr Gln Gly Al a Thr val Leu Gly Leu Phe Arg Val Thr 180 185 190 Pro Glu ile Pro Al a Gly Leu Pro Ser Pro Arg Ser Glu

195 200 205 <210> 10 <211> 150 <212> PRT <213> Artificial Sequence <220>

<223> : Synthetic construct <400> : 10 Pro Gly T rp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr 1 5 10 15 Phe Ser Pro Ala Leu Leu Val val Thr Glu Gly Asp Asn Ala Thr Phe 20 25 30 Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe val Leu Asn Trp Tyr 35 40 45 Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Al a Phe Pro Glu 50 55 60 Asp Arg Ser Gl n Pro Gly Gln Asp cys Arg Phe Arg Val Thr Gln Leu 65 70 75 80 Pro Asn Gly Arg Asp Phe His Met Ser val val Arg Ala Arg Arg Asn 85 90 95 Asp Ser Gly Thr Tyr Leu cys Gly Al a ile Ser Leu Ala Pro Lys Al a 100 105 110 Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg val Thr Glu Arg Arg 115 120 125

Page 11

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3105559_l.txt

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly 130 135 140

Gln Phe Gln Thr Leu Val

145 150 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic construct <400> 11

Glu Pro Lys Ser Cys Asp Lys

1 5 <210> 12 <211> 1032 <212> DNA <213> Artificial Sequence <220>

<223> Synthetic construct <400> 12

gctagcacca agggcccctc cgtgttccct ctggccccct ccagcaagtc cacctctggc 60 ggcaccgccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc 120 tggaactctg gcgctctgac ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc 180 ggcctgtact ccctgtcctc cgtcgtgacc gtgccctcca gctctctggg cacccagacc 240 tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa ggtggaaccc 300 aagtcctgcg acaagaccca cacctgtccc ccctgccctg cccctgagct cctgggaggc 360 cctagcgtgt tcctgttccc cccaaagccc aaggacaccc tgatgatctc ccggaccccc 420 gaagtgacct gcgtggtggt ggacgtgtcc cacgaggacc ctgaagtgaa gttcaattgg 480 tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ccagagagga acagtacaac 540 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaaa 600 gagtacaagt gcaaggtgtc caacaaggcc ctgcctgccc ccatcgaaaa gaccatctcc 660 aaggccaagg gccagccccg cgagcctcag gtgtacaccc tgccccctag ccgggaagag 720 atgaccaaga accaggtgtc cctgacctgt ctggtcaagg gcttctaccc ctccgatatc 780 gccgtggaat gggagtccaa cggccagccc gagaacaact acaagaccac cccccctgtg 840 ctggactccg acggctcatt cttcctgtac tccaagctga ccgtggacaa gtcccggtgg 900 cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 960 cagaagtccc tgtccctgag cccaggcaaa ggcggaggcg gatctggcgg cggaggatct 1020 ggtggcggat cc 1032

Page 12

2017201572 10 Mar 2017 <210> 13 <211> 425 <212> DNA <213> Artificial Sequence <220>

<223> Synthetic construct <400> 13

31O5559_l.txt

ggatccacca tccccccaca cgtgcagaaa tccgtgaaca acgacatgat cgtgaccgac 60 aacaacggcg ctgtgaagtt cccccagctg tgcaagttct gcgacgtgcg gttctctacc 120 tgcgacaacc agaaatcctg catgtccaac tgctccatca cctccatctg cgagaagccc 180 caggaagtgt gcgtcgccgt ctggcggaag aacgacgaga acatcaccct ggaaaccgtg 240 tgccacgacc ccaagctgcc ctaccacgac ttcatcctgg aagatgccgc ctcccccaag 300 tgcatcatga aggaaaagaa gaagcccggc gagactttct tcatgtgcag ctgctcctcc 360 gacgagtgca acgacaacat catcttctcc gaagagtaca acacctccaa ccccgactga 420

agctt 425 <210> 14 <211> 430 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct <400> 14 gcggccgcca tgaacttcgg cctgcggctg atcttcctgg tgctgaccct gaagggcgtg 60 cagtgcgagg tgcagctggt ggaatccggc ggaggcctgg tccagcctgg cggatctctg 120 agactgtcct gcgccgcctc cggcttcaac atcaaggaca cctacatcca ctgggtccga 180 caggcccctg gcaagggcct ggaatgggtg gcccggatct accccaccaa cggctacacc 240 agatacgccg actccgtgaa gggccggttc accatctccg ccgacacctc caagaacacc 300 gcctacctgc agatgaactc cctgcgggcc gaggacaccg ccgtgtacta ctgctccaga 360 tggggaggcg acggcttcta cgccatggac tactggggcc agggcaccct ggtcaccgtg 420 ctccgctagc 430 <210> 15 <211> 442 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 15 gcggccgcca tggaatccca gacccaggtg ctgatctccc tgctgttctg ggtgtccggc 60 acctgtggcg acatccagat gacccagtcc ccctccagcc tgtccgcctc tgtgggcgac 120 agagtgacca tcacctgtcg ggcctcccag gacgtgaaca ccgccgtggc ctggtatcag 180 cagaagcccg gcaaggcccc caagctgctg atctactccg cctccttcct gtactccggc 240 Page 13

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gtgccctccc ggttctccgg ctctagatcc ggcaccgact ttaccctgac catctccagc 300 ctgcagcccg aggacttcgc cacctactac tgccagcagc actacaccac cccccccacc 360 tttggccagg gcaccaaggt ggaaatcaag cggaccgtgg ccgctccctc cgtgttcatc 420 cccaccctcc gacgagcagc tg 442

<210> 16 <211> 1032 <212> DNA <213> Artificial Sequence <220>

<223> Synthetic construct <400> 16 gctagcacca agggcccctc cgtgtttccc ctggccccct ccagcaagtc cacctctggc 60 ggcaccgccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc 120 tggaactctg gcgctctgac ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc 180 ggcctgtact ccctgtcctc cgtcgtgacc gtgccctcca gctctctggg cacccagacc 240 tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagcg ggtggaaccc 300 aagtcctgcg acaagaccca cacctgtccc ccctgccctg cccctgaact gctgggaggc 360 ccttccgtgt tcctgttccc cccaaagccc aaggacaccc tgatgatctc ccggaccccc 420 gaagtgacct gcgtggtggt ggacgtgtcc cacgaggacc ctgaagtgaa gttcaattgg 480 tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ccagagagga acagtacaac 540 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaaa 600 gagtacaagt gcaaggtgtc caacaaggcc ctgcctgccc ccatcgaaaa gaccatctcc 660 aaggccaagg gccagccccg cgagcctcag gtgtacaccc tgcctcccag ccgggacgag 720 ctgaccaaga accaggtgtc cctgacctgt ctggtcaagg gcttctaccc ctccgatatc 780 gccgtggaat gggagtccaa cggccagccc gagaacaact acaagaccac cccccctgtg 840 ctggactccg acggctcatt cttcctgtac tccaagctga ccgtggacaa gtcccggtgg 900 cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 960 cagaagtccc tgtctctgag ccccggcaaa ggcggcggag gatctggcgg tggcggatca 1020 ggcggaggat cc 1032 <210> 17 <211> 427 <212> DNA <213> Artificial Sequence <220>

<223> Synthetic construct <400> 17 gcggccgcca tgaacttcgg cctgcggctg atcttcctgg tgctgaccct gaagggcgtg 60 cagtgccagg tgcagctgaa gcagtccgga cctggcctgg tgcagccttc ccagtccctg 120 tccatcacct gtaccgtgtc cggcttctcc ctgaccaact acggcgtgca ctgggtccga 180

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2017201572 10 Mar 2017 cagtccccag gcaagggcct ggaatggctg ggagtgattt ggagcggcgg caacaccgac 240 tacaacaccc ccttcacctc ccggctgtcc atcaacaagg acaactccaa gtcccaggtg 300 ttcttcaaga tgaactccct gcagtccaac gacaccgcca tctactactg cgccagagcc 360 ctgacctact atgactacga gttcgcctac tggggacagg gcaccctggt caccgtgtct 420 cgctagc 427 <210> 18 <211> 442 <212> DNA <213> Artificial Sequence <220>

<223> Synthetic construct <400> 18 gcggccgcca tggaatccca gacccaggtg ctgatctccc tgctgttctg ggtgtccggc 60 acctgtggcg acatcctgct gacccagtcc cccgtgatcc tgtccgtgtc tcctggcgag 120 cgggtgtcct tctcctgccg ggcctcccag tccatcggca ccaacatcca ctggtatcag 180 cagcggacca acggctcccc tcggctgctg attaagtacg cctccgagtc tatctccggc 240 atcccctccc ggttctccgg ctctggctcc ggcaccgact tcaccctgtc catcaactcc 300 gtggaatccg aggatatcgc cgactactac tgccagcaga acaacaactg gcccaccacc 360 ttcggcgctg gcaccaagct ggaactgaag cggaccgtgg ccgctccctc cgtgttcatc 420 cccaccctcc gacgagcagc tg 442 <210> 19 <211> 424 <212> DNA <213> Artificial Sequence <220>

<223> Synthetic construct <400> 19 gcggccgcca tgaacttcgg cctgcggctg atcttcctgg tgctgaccct gaagggcgtg 60 cagtgccagg tgcagctggt ggaatccggc ggaggcgtgg tgcagcctgg cagatccctg 120 agactgtcct gcgccgcctc cggcttcacc ttctccagct acaccatgca ctgggtccga 180 caggcccctg gcaagggcct ggaatgggtc accttcatca gctacgacgg caacaacaag 240 tactacgccg actccgtgaa gggccggttc accatctccc gggacaactc caagaacacc 300 ctgtacctgc agatgaactc cctgcgggcc gaggacaccg ccatctacta ctgcgcccgg 360 accggctggc tgggcccttt tgattactgg ggccagggca ccctggtcac cgtgtcctcc 420 tagc 424 <210> 20 <211> 445 <212> DNA <213> Artificial Sequence

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31O5559_l.txt <220>

<223> Synthetic construct <400> 20 gcggccgcca tggaatccca gacccaggtg ctgatctccc tgctgttctg ggtgtccggc 60 acctgtggcg agatcgtgct gacccagtcc cccggcaccc tgtctctgag ccctggcgag 120 agagccaccc tgtcctgcag agcctcccag tccgtgggct cctcctacct ggcttggtat 180 cagcagaagc ccggccaggc ccctcggctg ctgatctacg gcgctttctc tcgggccacc 240 ggcatccctg accggttctc tggctccggc tccggcaccg acttcaccct gaccatctcc 300 cggctggaac ccgaggactt cgccgtgtac tactgccagc agtacggctc ctccccctgg 360 acctttggcc agggcaccaa ggtggaaatc aagcggaccg tggccgctcc ctccgtgttc 420 cttcccaccc tccgacgagc agctg 445 <210> 21 <211> 1035 <212> DNA <213> Artificial Sequence <220>

<223> Synthetic construct <400> 21 gctagcacaa agggccctag tgtgtttcct ctggctccct cttccaaatc cacttctggt 60 ggcactgctg ctctgggatg cctggtgaag gattactttc ctgaacctgt gactgtctca 120 tggaactctg gtgctctgac ttctggtgtc cacactttcc ctgctgtgct gcagtctagt 180 ggactgtact ctctgtcatc tgtggtcact gtgccctctt catctctggg aacccagacc 240 tacatttgta atgtgaacca caaaccatcc aacactaaag tggacaaaaa agccgaaccc 300 aaatcctgtg acaaaaccca cacctgccca ccttgtcctg cccctgaact gctgggagga 360 ccttctgtgt ttctgttccc accaaaacca aaagataccc tgatgatctc tagaacccct 420 gaggtgacat gtgtggtggt ggatgtgtct catgaggacc ctgaggtcaa atttaattgg 480 tacgtcgatg gagtggaagt ccacaatgcc aaaaccaagc ctagagagga acagtacaat 540 tcaacctaca gagtcgtcag tgtgctgact gtgctgcatc aggattggct gaatggcaag 600 gaatacaagt gtaaagtctc aaacaaggcc ctgcctgctc caattgagaa aacaatctca 660 aaggccaagg gacagcctag ggaaccccag gtctacaccc tgccaccttc acgcgacgaa 720 ctgaccaaaa accaggtgtc cctgacatgc ctggtcaaag gcttctaccc ttctgacatt 780 gctgtggagt gggagtcaaa tggacagcct gagaacaact acaaaacaac cccccctgtg 840 ctggattctg atggctcttt ctttctgtac tccaaactga ctgtggacaa gtctagatgg 900 cagcagggga atgtcttttc ttgctctgtc atgcatgagg ctctgcataa ccactacact 960 cagaaatccc tgtctctgtc tcccgggaaa ggcggcggag gatctggcgg aggcggttct 1020 ggtggtggcg gatcc 1035 <210> 22 <211> 435

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31O5559_l.txt <212> DNA <213> Artificial Sequence <220>

<223> Synthetic construct <400> 22 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60 cagtgtcagg tgcagctgca gcagcctggt gccgagctcg tgaaacctgg cgcctccgtg 120 aagatgtcct gcaaggcctc cggctacacc ttcaccagct acaacatgca ctgggtcaag 180 cagacccccg gcagaggcct ggaatggatc ggcgctatct accccggcaa cggcgacacc 240 tcctacaacc agaagttcaa gggcaaggcc accctgaccg ccgacaagtc ctcttccacc 300 gcctacatgc agctgtcctc cctgacctcc gaggactccg ccgtgtacta ctgcgcccgg 360 tctacctact acggcggcga ctggtacttc aacgtgtggg gcgctggcac caccgtgacc 420 gtgtctgctg ctagc 435 <210> 23 <211> 405 <212> DNA <213> Artificial Sequence <220>

<223> Synthetic construct <400> 23 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60 cagtgtcaga tcgtgctgtc ccagtcccct gccatcctgt ctgctagccc tggcgagaaa 120 gtgacaatga cctgccgggc ctcctcctcc gtgtcctaca tccactggtt ccagcagaag 180 cccggctcca gccccaagcc ttggatctac gccacctcca acctggcctc tggcgtgcca 240 gtgcggtttt ccggctctgg ctctggcacc tcctactccc tgaccatctc tcgggtggaa 300 gccgaggatg ccgccaccta ctactgccag cagtggacca gcaacccccc cacatttggc 360 ggaggcacca agctggaaat caagcggacc gtggcggcgc cctct 405 <210> 24 <211> 631 <212> DNA <213> Artificial Sequence <220>

<223> Synthetic construct <400> 24 ggatccgcct gtccttgggc cgtgtccggc gctagagcct ctcctggctc tgccgcctcc 60 cccagactga gagagggccc tgagctgtcc cctgacgatc ctgccggcct gctggacctg 120 agacagggca tgtttgccca gctggtggcc cagaacgtgc tgctgatcga cggccccctg 180 tcctggtact ctgatcctgg cctggccggc gtgtccctga ccggcggact gtcctacaaa 240 gaggacacca aagaactggt ggtggccaag gctggcgtgt actacgtgtt ctttcagctg 300 gaactgcggc gggtggtggc cggcgagggc tctggatctg tgtccctggc cctgcatctg 360

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31O5559_l.txt cagcccctga gatctgccgc tggcgccgct gctctggccc tgacagtgga tctgcctcct 420 gcctcctccg aggcccggaa ctccgcattc gggtttcagg gccggctgct gcacctgtct 480 gctggccaga gactgggagt gcatctgcac accgaggcca gagccagaca cgcctggcag 540 ctgacccagg gcgctaccgt gctgggcctg ttcagagtga cccccgagat cccagccggc 600 ctgcccagcc ctagatccga gtgataagct t 631 <210> 25 <211> 1458 <212> DNA <213> Artificial Sequence <220>

<223> Synthetic construct <400> 25 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60 cagtgtcagg tgcagctgca ggaatctggc cctggactcg tgcggccttc ccaaaccctg 120 tctctgacct gtaccgtgtc cggctactcc atcacctccg accacgcctg gtcttgggtg 180 cgacagcctc ctggcagagg cctggaatgg atcggctaca tctcctactc cggcatcacc 240 acctacaacc ccagcctgaa gtccagagtg accatgctgc gggacacctc caagaaccag 300 ttctccctgc ggctgtcctc cgtgaccgct gctgataccg ccgtgtacta ctgcgccaga 360 tctctggcca ggaccaccgc catggattac tggggccagg gctccctcgt gaccgtgtcc 420 tctgctagca ccaagggccc ctccgtgttc cctctggccc cttcctctaa atctacctct 480 ggcggcaccg ccgctctggg ctgcctcgtg aaggactact tccccgagcc cgtgacagtg 540 tcttggaact ctggcgccct gacctccggc gtgcacacct ttccagctgt gctgcagtcc 600 tccggcctgt actccctgtc cagcgtcgtg actgtgccct cctcatctct gggcacccag 660 acctacatct gcaacgtgaa ccacaagccc tccaacacca aggtggacaa gaaggtggaa 720 cccaagtcct gcgacaagac ccacacctgt cccccttgtc ctgcccctga actgctgggc 780 ggaccctctg tgttcctgtt cccaccaaaa ccgaaagaca ccctgatgat ctcccggacc 840 cccgaagtga cctgcgtggt ggtggatgtg tcccacgagg accctgaagt gaagttcaat 900 tggtacgtgg acggcgtgga agtgcacaac gccaagacca agcctagaga ggaacagtac 960 aactccacct accgggtggt gtccgtgctg accgtgctgc accaggattg gctgaacggc 1020 aaagagtaca agtgcaaggt gtccaacaag gccctgcctg cccccatcga aaagaccatc 1080 tccaaggcca agggccagcc acgggaaccc caggtgtaca cactgccccc tagccgcgac 1140 gagctgacca agaatcaggt gtccctgaca tgcctcgtga aaggcttcta cccctccgat 1200 atcgccgtgg aatgggagtc caacggccag cctgagaaca actacaagac caccccccct 1260 gtgctggact ccgacggctc attcttcctg tactcaaagc tgacagtgga caagtcccgg 1320 tggcagcagg gcaacgtgtt ctcctgctcc gtgatgcacg aggccctgca caaccactac 1380 acccagaagt ccctgtccct gagccccggg aaaggcggcg gaggatctgg cggaggcggt 1440 tctggtggtg gcggatcc 1458

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2017201572 10 Mar 2017 <210> 26 <211> 405 <212> DNA <213> Artificial Sequence <220>

<223> Synthetic construct <400> .26 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60 cagtgtgaca tccagatgac ccagtccccc tccagcctgt ctgcctctgt gggcgacaga 120 gtgaccatca cctgtcgggc ctcccaggac atctcctcct acctgaactg gtatcagcag 180 aagcccggca aggcccccaa gctgctgatc tactacacct cccggctgca ctccggcgtg 240 ccctctagat tttccggctc tggctccggc accgacttta ccttcaccat cagctccctg 300 cagcccgagg atatcgccac ctactactgc cagcaaggca acaccctgcc ctacaccttt 360 ggccagggca ccaaggtgga aatcaagcgg accgtggcgg cgccc 405 <210> 27 <211> 1455 <212> DNA <213> Artificial Sequence <220>

<223> Synthetic construct <400> 27 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc 60 cagtgtcagg tgcagctgca gcagtgggga gctggactgc tgaagccctc cgagacactg 120 tctctgacct gcgctgtgta cggcggctcc ttctccggct actactggtc ctggattcgg 180 cagtcccctg agaagggcct ggaatggatc ggcgagatca accacggcgg ctacgtgacc 240 tacaacccca gcctggaatc cagagtgacc atctccgtgg acacctccaa gaaccagttc 300 tccctgaagc tgtcctccgt gaccgccgct gataccgccg tgtactactg cgccagagac 360 tacggccctg gcaactacga ctggtacttc gacctgtggg gcagaggcac cctcgtgacc 420 gtgtcctctg ctagcaccaa gggcccctcc gtgtttcctc tggccccttg ctcacgctcc 480 acctccgaat ctaccgccgc tctgggctgc ctcgtgaagg actacttccc cgagcccgtg 540 actgtgtctt ggaactctgg cgccctgacc tccggcgtgc acacctttcc agctgtgctg 600 cagtcctccg gcctgtactc cctgtccagc gtcgtgacag tgccctccag ctctctgggc 660 accaagacct acacctgtaa cgtggaccac aagccctcca acaccaaggt ggacaagcgg 720 gtggaatcta aatacggccc tccctgccct ccttgcccag cccctgaatt tctgggcgga 780 ccttccgtgt tcctgttccc cccaaaaccc aaggacaccc tgatgatctc ccggaccccc 840 gaagtgacct gcgtggtggt ggatgtgtcc caggaagatc ccgaggtgca gttcaattgg 900 tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ctagagagga acagttcaac 960 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggattggct gaacggcaaa 1020

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3105559_l.txt gagtacaagt gcaaggtgtc caacaagggc ctgcccagct ccatcgaaaa gaccatcagc aaggccaagg gccagccccg ggaaccccag gtgtacacac tgcctccaag ccaggaagag atgaccaaga atcaggtgtc cctgacctgt ctcgtgaaag gcttctaccc ctccgatatc gccgtggaat gggagtccaa cggccagcct gagaacaact acaagaccac cccccctgtg ctggactccg acggcagctt cttcctgtac tctcgcctga ccgtggacaa gtcccggtgg caggaaggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc cagaagtccc tgtccctgtc tctggggaaa ggcggcggag gatctggcgg aggcggttct ggtggtggcg gatcc <210> 28 <211> 411 <212> DNA <213> Artificial Sequence <220>

<223> Synthetic construct <400> 28 gcggccgcca tgaattttgg actgaggctg attttcctgg tgctgaccct gaaaggcgtc cagtgtgaga tcgtgctgac ccagtctcct gccaccctgt ctctgagccc tggcgagaga gctaccctgt cctgccgtgc ctcccaatcc gtgtcctctt acctggcctg gtatcagcaa aagcccggcc aggctccccg gctgctgatc tacgatgcct ccaatagagc caccggcatc cctgccagat tctccggctc tggctctggc accgacttta ccctgaccat ctcctctctg gaacccgagg acttcgccgt gtactactgc cagcagcggt ccaactggcc tcccgccctg acatttggcg gaggcaccaa ggtggaaatc aagcggaccg tggcggcgcc c

1080

1140

1200

1260

1320

1380

1440

1455

120

180

240

300

360

411 <210> 29 <211> 449 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic construct <400> 29

Gln 1 Val Gln Leu Val 5 Gln Ser Gly Ala Glu 10 Val Lys Lys Pro Gly 15 Glu Thr Val Lys Ile Ser cys Lys Ala Ser Asp Tyr Thr Phe Thr Tyr Tyr 20 25 30 Gly Met Asn T rp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asp Thr Thr Thr Gly Glu Pro Thr Tyr Al a Gln Lys Phe 50 55 60 Gln Gly Arg ile Al a Phe Ser Leu Glu Thr Ser Al a Ser Thr Ala Tyr 65 70 75 80 Page 20

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31O5559_l.txt

Leu Gin Ile Lys Ser 85 Leu Lys Ser Glu Asp Thr Ala Thr Tyr 90 Phe 95 cys Al a Arg Arg Gly Pro Tyr Asn Trp Tyr Phe Asp Val T rp Gly Gin Gly 100 105 110 Thr Thr val Thr val Ser Ser Ala Ser Thr Lys Gly Pro Ser val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Al a Al a Leu 130 135 140 Gly Cys Leu val Lys Asp Tyr Phe Pro Glu Pro Val Thr val Ser Trp 145 150 155 160 Asn Ser Gly Al a Leu Thr Ser Gly Val His Thr Phe Pro Ala val Leu 165 170 175 Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gin Thr Tyr ile Cys Asn Val Asn Hi s Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys val Glu Pro Lys Ser cys Asp Lys 210 215 220 Thr His Thr cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp val Ser Hi s Glu Asp 260 265 270 Pro Glu val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu val Hi s Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg val 290 295 300 val Ser Val Leu Thr Val Leu Hi s Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys cys Lys Val Ser Asn Lys Al a Leu Pro Al a Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Al a Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350

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Leu Pro Pro Ser Arg Asp Glu 355 Leu Thr 360 Lys Asn Gln Val 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp ile Ala val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser val Met Hi s Glu 420 425 430 Al a Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 30 <211> 219 <212> PRT <213> , Artificial Sequence <220> <223> Synthetic construct <400> 30 Asp lie Val Met Thr Gln Ser Pro Leu Ser Val Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu lie Tyr Arg Met Ser Asn Leu val Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp val Gly Val Tyr Tyr Cys Leu Gln Hi s 85 90 95 Leu Glu Tyr Pro Phe Thr Phe Gly Pro Gly Thr Lys Leu Glu Leu Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser val Phe ile Phe Pro Pro Ser Asp Glu 115 120 125

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Gin Leu 130 Lys Ser Gly Thr Ala 135 Ser val Val cys Leu 140 Leu Asn Asn Phe Tyr Pro Arg Glu Al a Lys Val Gin Trp Lys Val Asp Asn Al a Leu Gin 145 150 155 160 Ser Gly Asn Ser Gin Glu Ser val Thr Glu Gin Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys Hi s Lys Val Tyr Al a cys Glu Val Thr Hi s Gl n Gly Leu Ser Ser 195 200 205 Pro val Thr Lys Ser Phe Asn Arg Gly Glu cys 210 215 <210> 31 <211> 460 <212> 1 PRT <213> , Artificial Sequence <220> <223> : Synthetic construct <400> 31 Glu val Gin Leu Val Gin Ser Gly Ala Glu val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys val Ser Cys Lys Ala Ser Gly Gly Thr phe Ser Ser Tyr 20 25 30 Ala lie Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Gly lie lie Pro lie Phe Gly Thr Ala Asn Tyr Ala Gin Lys Phe 50 55 60 Gin Gly Arg Val Thr lie Thr Ala Asp Lys Ser Thr Ser Thr Al a Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala val Tyr Tyr cys 85 90 95 Ala Arg Ala Pro Leu Arg Phe Leu Glu Trp Ser Thr Gin Asp Hi s Tyr 100 105 110 Tyr Tyr Tyr Tyr Met Asp val Trp Gly Lys Gly Thr Thr Val Thr Val 115 120 125 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Al a Pro Ser Page 23

2017201572 10 Mar 2017

310555 ,9_1. txt 130 135 140 Ser Lys Ser Thr Ser Gly Gly Thr Al a Ala Leu Gly cys Leu val Lys 145 150 155 160 Asp Tyr Phe Pro Glu Pro val Thr val Ser Trp Asn Ser Gly Ala Leu 165 170 175 Thr Ser Gly val Hi s Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 180 185 190 Tyr Ser Leu Ser Ser Val val Thr val Pro Ser Ser Ser Leu Gly Thr 195 200 205 Gln Thr Tyr ile cys Asn val Asn Hi s Lys Pro Ser Asn Thr Lys val 210 215 220 Asp Lys Lys val Glu Pro Lys Ser cys Asp Lys Thr Hi s Thr cys Pro 225 230 235 240 Pro cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 245 250 255 pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu val 260 265 270 Thr cys val Val Val ASP val Ser His Glu Asp Pro Glu Val Lys Phe 275 280 285 Asn Trp Tyr Val Asp Gly val Glu Val Hi s Asn Al a Lys Thr Lys Pro 290 295 300 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg val Val Ser val Leu Thr 305 310 315 320 Val Leu Hi s Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys cys Lys val 325 330 335 Ser Asn Lys Al a Leu Pro Al a Pro Ile Glu Lys Thr lie Ser Lys Al a 340 345 350 Lys Gly Gln Pro Arg Glu Pro Gln val Tyr Thr Leu Pro Pro Ser Arg 355 360 365 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu val Lys Gly 370 375 380 Phe Tyr Pro Ser Asp ile Al a val Glu Trp Glu Ser Asn Gly Gln Pro 385 390 395 400 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 405 410 415

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Phe Phe Leu Tyr Ser Lys 420 Leu Thr Val 425 Asp Lys Ser Arg Trp 430 Gln Gln Gly Asn Val Phe Ser Cys Ser val Met Hi s Glu Ala Leu Hi s Asn Hi s 435 440 445 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455 460 <210> 32 <211> 213 <212> PRT <213> Artificial Sequence <220>

<223> : Synthetic construct <400> 32 Ser Ser Glu Leu Thr Gln Asp Pro Al a Val Ser val Al a Leu Gly Gln 1 5 10 15 Thr Val Arg lie Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Al a 20 25 30 Thr Trp Tyr Gln Gln Lys Pro Gly Gln Al a Pro Ile Leu val lie Tyr 35 40 45 Gly Glu Asn Lys Arg pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Al a Ser Leu Thr ile Thr Gly Al a Gln Al a Glu 65 70 75 80 Asp Glu Al a Asp Tyr Tyr cys Lys Ser Arg Asp Gly Ser Gly Gln Hi s 85 90 95 Leu val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Al a Al a Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Al a Thr Leu Val cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 13 5 140 Ala Val Thr val Ala T rp Lys Al a Asp Ser Ser Pro val Lys Al a Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Al a Al a 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser Hi s Arg Ser

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3105559_l.txt

180 185

Tyr Ser Cys Gin Val Thr His Glu Gly Ser Thr Val 195 200

Ala Pro Ala Glu Cys 210 <210> 33 <211> 449 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic construct <400> 33

190

Glu Lys Thr Val 205

Gin Val 1 Gin Leu Gin Gin 5 Ser Gly Ala Glu 10 Val Lys Lys Phe Gly 15 Ala Ser Val Lys Val Ser cys Glu Ala Ser Gly Tyr Thr Phe Pro Ser Tyr 20 25 30 Val Leu His Trp Val Lys Gin Al a Pro Gly Gin Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Gin Thr Asn Lys Lys Phe 50 55 60 Lys Gly Lys Al a Thr Leu Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Al a Val Tyr Tyr Cys 85 90 95 Al a Arg Gly Phe Gly Gly Ser Tyr Gly Phe Ala Tyr Asn Gly Gin Gly 100 105 110 Thr Leu Val Thr val Ser Ser Al a Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Al a Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr val Ser Trp 145 150 155 160 Asn Ser Gly Al a Leu Thr Ser Gly Val Asn Thr Phe Pro Al a val Leu 165 170 175 Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser val Val Thr Val Pro Ser 180 185 190

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31O5559_l.txt

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn val Asn 205 Hi s Lys Pro 195 200 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser cys Asp Lys 210 215 220 Thr Hi s Thr cys Pro Pro cys Pro Al a Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Asn ile Ser 245 250 255 Arg Thr Pro Glu Val Thr cys Val val Val Asp Val Ser Hi s Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr val Asp Gly Val Glu Val Hi s Asn 275 280 285 Al a Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg val 290 295 300 Val Ser Val Leu Thr val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys val Ser Asn Lys Al a Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Al a Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Asn Hi s Glu 420 425 430 Al a Leu Hi s Asn Hi s Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445

Lys <210> 34

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3105559_l.txt

2017201572 10 Mar 2017 <211> 215 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic construct <400> 34

Asp 1 ile Gin Leu Thr 5 Gin Ser Pro Asp Arg Val Thr 20 Met Thr Cys Ser Tyr Leu Tyr 35 Trp Tyr Gin Gin Lys 40 Ile Tyr 50 Ser Thr Ser Asn Leu 55 Ala Gly 65 Ser Gly Ser Gly Thr 70 Asp Phe Pro Glu Asp Ser Al a 85 Ser Tyr Phe Tyr Thr Phe Gly 100 Gly Gly Thr Arg Ala Pro Ser 115 Val Phe Ile Phe Pro 120 Gly Thr 130 Al a Ser Val Val cys 135 Leu Val 145 Gin Trp Lys Val Asp 150 Asn Al a Ser Val Thr Glu Gin 165 Asp Ser Lys Thr Leu Thr Leu 180 Ser Pro Arg Lys Tyr Ala Cys 195 Glu val Thr Hi s Gin 200 Ser Phe 210 Asn Arg Gly Glu cys 215 <210> <211> <212> <213> . 35 450 PRT Artificial Sequence

Ser Ser 10 Leu Ser Al a Ser Val 15 Gly Ala 25 Ser Ser Ser val Ser 30 Ser Ser Pro Gly Lys Ala Pro 45 Lys Leu Trp Ser Gly Val Pro 60 Al a Arg Phe Ser Thr Leu Thr 75 Ile Ser Ser Leu Gin 80 Cys Hi s 90 Gin Trp Asn Arg Tyr 95 Pro Leu 105 Glu ile Lys Arg Thr 110 val Al a Pro Ser Asp Glu Gin 125 Leu Lys Ser Leu Asn Asn Phe 140 Tyr Glu Al a Lys Leu Gin Ser 155 Gly Asn Ser Gin Glu 160 Asp Ser 170 Thr Tyr Ser Leu Ser 175 Ser Al a 185 Asp Tyr Glu Lys Hi s 190 Lys val Gly Leu Ser Ser Pro 205 Val Thr Lys

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<223> Synthetic construct <400> 35

31O5559_l.txt

Glu 1 val Gin Leu Leu 5 Glu Ser Gly Gly Asp 10 Leu val Gin Pro Gly 15 Gly Ser Leu Arg Leu Ser Cys Al a Al a Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Al a Met Ser T rp Val Arg Gin Al a Pro Gly Lys Gly Leu Glu T rp Val 35 40 45 Ser Ala lie Thr Pro Ser Gly Gly Ser Thr Asn Tyr Al a Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Gin Asn Thr Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg val Glu Asp Thr Al a Val Tyr lie cys 85 90 95 Gly Arg Val Pro Tyr Arg Ser Thr Trp Tyr Pro Leu Tyr Trp Gly Gin 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Al a Ser Thr Lys Gly Pro Ser val 115 120 125 Phe Pro Leu Al a Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr val Ser 145 150 155 160 T rp Asn Ser Gly Ala Leu Thr Ser Gly val Hi s Thr Phe Pro Al a Val 165 170 175 Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr Hi s Thr cys Pro Pro cys Pro Al a Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie 245 250 255

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Ser Arg Thr Pro Glu Val 260 Thr Cys val 265 Val val Asp Val Ser 270 Hi s Glu Asp Pro Glu val Lys Phe Asn Trp Tyr val Asp Gly Val Glu Val Hi s 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 val Val Ser val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys val Ser Asn Lys Al a Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr ile Ser Lys Al a Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr cys Leu val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn val Phe Ser cys Ser Val Met Hi s 420 425 430 Glu Ala Leu His Asn Hi s Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys 450 <210> 36 <211> 214 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic construct <400> 36

Asp Ile Gl n Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg val Thr Ile Thr Cys Arg Al a Ser Gln Asp Ile Ser Asn Tyr 20 25 30

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31O5559_l.txt

Leu Ala Trp 35 Phe Gin Gin Lys Pro Gly Lys Ala Pro Lys Ser Leu lie 40 45 Tyr Ala Al a Ser Ser Leu Hi s Ser Lys Val Pro Thr Gin Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr cys Leu Gin Tyr Ser Thr Tyr Pro Ile 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125 Thr Ala Ser val val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys val Gin Trp Lys val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin 145 150 155 160 Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys val Tyr 180 185 190 Ala Cys Glu Val Thr Hi s Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu cys 210 <210> 37 <211> 450 <212> PRT <213> 1 Homo sapi ens <400> 37 Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Al a Al a Ser Gly Phe Asn lie Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gin Al a Pro Gly Lys Gly Leu Glu Trp val 35 40 45

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Al a Arg 50 lie Tyr Pro Thr Asn 55 Gly Tyr Thr Arg Tyr Ala Asp Ser 60 Val Lys Gly Arg Phe Thr lie Ser Al a Asp Thr Ser Lys Asn Thr Al a Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Al a Gl u Asp Thr Ala Val Tyr Tyr cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Al a Met Asp Tyr T rp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser val 115 120 125 Phe Pro Leu Al a Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Al a Al a 130 135 140 Leu Gly Cys Leu val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Al a Leu Thr Ser Gly Val His Thr Phe Pro Al a Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr lie Cys Asn Val Asn Hi s Lys 195 200 205 Pro Ser Asn Thr Lys val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr Hi s Thr cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie 245 250 255 Ser Arg Thr Pro Glu val Thr cys Val Val val Asp Val Ser Hi s Glu 260 265 270 Asp Pro Gl u Val Lys Phe Asn T rp Tyr val Asp Gly Val Glu Val Hi s 275 280 285 Asn Al a Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 val Val Ser Val Leu Thr Val Leu Hi s Gln Asp T rp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys cys Lys Val Ser Asn Lys Ala Leu Pro Al a Pro lie Glu Page 32

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325 330 335 Lys Thr ile Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu 355 360 365 Thr Cys Leu val Lys Gly Phe Tyr Pro Ser Asp lie Al a val Glu T rp 370 375 380 Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser val Met Hi s 420 425 430 Glu Al a Leu Hi s Asn Hi s Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 38 <211> 449 <212> 1 PRT <213> 1 Homo sap^- i ens <4oo> : 38 Gin Val Gin Leu Lys Gin Ser Gly Pro Gly Leu Val Gin Pro Ser Gl n 1 5 10 15 Ser Leu Ser Ile Thr cys Thr val Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val Hi s T rp Val Arg Gin Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly val Ile T rp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gin Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gin Ser Asn Asp Thr Al a Ile Tyr Tyr Cys Al a 85 90 95 Arg Al a Leu Thr Tyr Tyr Asp Tyr Glu Phe Al a Tyr Trp Gly Gin Gly 100 105 110

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Thr Leu val 115 Thr Val Ser Al a Al a 120 Ser Thr Lys Gly Pro 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Al a Leu 130 135 140 Gly cys Leu val Lys Asp Tyr Phe Pro Glu Pro val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Al a Leu Thr Ser Gly val Hi s Thr Phe Pro Ala val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr lie Cys Asn val Asn Hi s Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg val Glu Pro Lys Ser cys Asp Lys 210 215 220 Thr Hi s Thr cys Pro Pro Cys Pro Al a Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Hi s Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val Hi s Asn 275 280 285 Al a Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg val 290 295 300 Val Ser Val Leu Thr val Leu Hi s Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Al a Leu Pro Al a Pro Ile Glu Lys 325 330 335 Thr lie Ser Lys Al a Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 cys Leu Val Lys Gly Phe Tyr Pro Ser Asp ile Al a val Glu T rp Glu 370 375 380

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31O5559_l.txt

Ser 385 Asn Gly Gin Pro Glu 390 Asn Asn Tyr Lys Thr 395 Thr Pro Pro Val Leu 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gin Gin Gly Asn val Phe Ser cys Ser Val Met Hi s Glu 420 425 430 Al a Leu Hi s Asn Hi s Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 39 <211> 448 <212> PRT <213> Homo sapi ens <400> 39 Gin val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Al a Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met Hi s T rp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Phe lie Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala lie Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gin Gly Thr 100 105 110 Leu val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe pro 115 120 125 Leu Al a Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Al a Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr val Ser T rp Asn 145 150 155 160 Ser Gly Al a Leu Thr Ser Gly val Hi s Thr Phe Pro Al a val Leu Gin 165 170 175

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31O5559_l.txt

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 185 Val Thr Val Pro 190 Ser Ser 180 Ser Leu Gly Thr Gl n Thr Tyr ile cys Asn Val Asn Hi s Lys Pro Ser 195 200 205 Asn Thr Lys val Asp Lys Arg Val Glu Pro Lys Ser cys Asp Lys Thr 210 215 220 Hi s Thr Cys Pro Pro cys Pro Al a Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg 245 250 255 Thr Pro Glu val Thr cys val val Val Asp val Ser Hi s Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr val Asp Gly val Glu Val Hi s Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu Hi s Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys cys Lys val Ser Asn Lys Al a Leu Pro Al a Pro Ile Glu Lys Thr 325 330 335 lie Ser Lys Al a Lys Gly Gln Pro Arg Glu Pro Gln val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg T rp Gln Gln Gly Asn val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu Hi s Asn Hi s Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445

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